Biodegradable lipids for the delivery of active agents

ABSTRACT

The present invention relates to a cationic lipid having one or more biodegradable groups located in a lipidic moiety (e.g., a hydrophobic chain) of the cationic lipid. These cationic lipids may be incorporated into a lipid particle for delivering an active agent, such as a nucleic acid. The invention also relates to lipid particles comprising a neutral lipid, a lipid capable of reducing aggregation, a cationic lipid of the present invention, and optionally, a sterol. The lipid particle may further include a therapeutic agent such as a nucleic acid.

This application is a continuation of U.S. patent application Ser. No.13/708,383, filed Dec. 7, 2012, which claims the benefit of U.S.Provisional Application No. 61/568,133, filed Dec. 7, 2011, and U.S.Provisional Application No. 61/623,274, filed Apr. 12, 2012, each ofwhich is hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to biodegradable lipids and to their usefor the delivery of active agents such as nucleic acids.

BACKGROUND

Therapeutic nucleic acids include, e.g., small interfering RNA (siRNA),micro RNA (miRNA), antisense oligonucleotides, ribozymes, plasmids,immune stimulating nucleic acids, antisense, antagomir, antimir,microRNA mimic, supermir, U1 adaptor, and aptamer. In the case of siRNAor miRNA, these nucleic acids can down-regulate intracellular levels ofspecific proteins through a process termed RNA interference (RNAi). Thetherapeutic applications of RNAi are extremely broad, since siRNA andmiRNA constructs can be synthesized with any nucleotide sequencedirected against a target protein. To date, siRNA constructs have shownthe ability to specifically down-regulate target proteins in both invitro and in vivo models. In addition, siRNA constructs are currentlybeing evaluated in clinical studies.

However, two problems currently faced by siRNA or miRNA constructs are,first, their susceptibility to nuclease digestion in plasma and, second,their limited ability to gain access to the intracellular compartmentwhere they can bind the protein RISC when administered systemically asthe free siRNA or miRNA. Lipid nanoparticles formed from cationic lipidswith other lipid components, such as cholesterol and PEG lipids, andoligonucleotides (such as siRNA and miRNA) have been used to facilitatethe cellular uptake of the oligonucleotides.

There remains a need for improved cationic lipids and lipidnanoparticles for the delivery of oligonucleotides. Preferably, theselipid nanoparticles would provide high drug:lipid ratios, protect thenucleic acid from degradation and clearance in serum, be suitable forsystemic delivery, and provide intracellular delivery of the nucleicacid. In addition, these lipid-nucleic acid particles should bewell-tolerated and provide an adequate therapeutic index, such thatpatient treatment at an effective dose of the nucleic acid is notassociated with significant toxicity and/or risk to the patient.

SUMMARY

The present invention relates to a cationic lipid and PEG lipid suitablefor forming nucleic acid-lipid particles. Each of the cationic and PEGlipids of the present invention includes one or more biodegradablegroups. The biodegradable groups are located in a lipidic moiety (e.g.,a hydrophobic chain) of the cationic or PEG lipid. These cationic andPEG lipids may be incorporated into a lipid particle for delivering anactive agent, such as a nucleic acid (e.g., an siRNA). The incorporationof the biodegradable group(s) into the lipid results in fastermetabolism and removal of the lipid from the body following delivery ofthe active agent to a target area. As a result, these lipids have lowertoxicity than similar lipids without the biodegradable groups.

In one embodiment, the cationic lipid is a compound of formula (I),which has a branched alkyl at the alpha position adjacent to thebiodegradable group (between the biodegradable group and the tertiarycarbon):

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof),wherein

R′ is absent, hydrogen, or alkyl (e.g., C₁-C₄ alkyl);

with respect to R¹ and R²,

-   -   (i) R¹ and R² are each, independently, optionally substituted        alkyl, alkenyl, alkynyl, cycloalkylalkyl, heterocycle, or R¹⁰;    -   (ii) R¹ and R², together with the nitrogen atom to which they        are attached, form an optionally substituted heterocylic ring;        or    -   (iii) one of R¹ and R² is optionally substituted alkyl, alkenyl,        alkynyl, cycloalkyl, cycloalkylalkyl, or heterocycle, and the        other forms a 4-10 member heterocyclic ring or heteroaryl (e.g.,        a 6-member ring) with (a) the adjacent nitrogen atom and (b) the        (R)_(a) group adjacent to the nitrogen atom;

each occurrence of R is, independently, —(CR³R⁴)—;

each occurrence of R³ and R⁴ are, independently H, halogen, OH, alkyl,alkoxy, —NH₂, R¹⁰, alkylamino, or dialkylamino (in one preferredembodiment, each occurrence of R³ and R⁴ are, independently H or C₁-C₄alkyl);

each occurrence of R¹⁰ is independently selected from PEG and polymersbased on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol),poly(glycerol), poly(N-vinylpyrrolidone),poly[N-(2-hydroxypropyl)methacrylamide] and poly(amino acid)s, wherein(i) the PEG or polymer is linear or branched, (ii) the PEG or polymer ispolymerized by n subunits, (iii) n is a number-averaged degree ofpolymerization between 10 and 200 units, and (iv) wherein the compoundof formula has at most two R¹⁰ groups (preferably at most one R¹⁰group);

the dashed line to Q is absent or a bond;

-   -   when the dashed line to Q is absent then Q is absent or is —O—,        —NH—, —S—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)N(R⁴)—, —N(R⁵)C(O)—,        —S—S—, —OC(O)O—, —O—N═C(R⁵)—, —C(R⁵)═N—O—, —OC(O)N(R⁵)—,        —N(R⁵)C(O)N(R⁵)—, —N(R⁵)C(O)O—, —C(O)S—, —C(S)O— or        —C(R⁵)═N—O—C(O)—; or

when the dashed line to Q is a bond then (i) b is 0 and (ii) Q and thetertiary carbon adjacent to it (C*) form a substituted or unsubstituted,mono- or bi-cyclic heterocyclic group having from 5 to 10 ring atoms(e.g., the heteroatoms in the heterocyclic group are selected from O andS, preferably O);

each occurrence of R⁵ is, independently, H or alkyl (e.g. C₁-C₄ alkyl);

X and Y are each, independently, alkylene or alkenylene (e.g., C₄ to C₂₀alkylene or C₄ to C₂₀ alkenylene);

M¹ and M² are each, independently, a biodegradable group (e.g., —OC(O)—,—C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O—, —S—S—, —C(R⁵)═N—,—N═C(R⁵)—, —C(R⁵)═N—O—, —O—N═C(R⁵)—, —C(O)(NR⁵)—, —N(R⁵)C(O)—,—C(S)(NR⁵)—, —N(R⁵)C(O)—, —N(R⁵)C(O)N(R⁵)—, —OC(O)O—, —OSi(R⁵)₂O—,—C(O)(CR³R⁴)C(O)O—, —OC(O)(CR³R⁴)C(O)—, or

(wherein R¹¹ is a C₂-C₈ alkyl or alkenyl));

each occurrence of R^(z) is, independently, C₁-C₈ alkyl (e.g., methyl,ethyl, isopropyl, n-butyl, n-pentyl, or n-hexyl);

a is 1, 2, 3, 4, 5 or 6;

b is 0, 1, 2, or 3; and

Z¹ and Z² are each, independently, C₈-C₁₄ alkyl or C₈-C₁₄ alkenyl,wherein the alkenyl group may optionally be substituted with one or twofluorine atoms at the alpha position to a double bond which is betweenthe double bond and the terminus of Z¹ or Z² (e.g.,

The R′R¹R²N—(R)_(a)-Q-(R)_(b)— group can be any of the head groupsdescribed herein, including those shown in Table 1 below, and saltsthereof. In one preferred embodiment, R′R¹R²N—(R)_(a)-Q-(R)_(b)— is(CH₃)₂N—(CH₂)₃—C(O)O—, (CH₃)₂N—(CH₂)₂—NH—C(O)O—,(CH₃)₂N—(CH₂)₂—OC(O)—NH—, or (CH₃)₂N—(CH₂)₃—C(CH₃)═N—O—.

In one embodiment, R¹ and R² are both alkyl (e.g., methyl).

In a further embodiment, a is 3. In another embodiment, b is 0.

In a further embodiment, a is 3, b is 0 and R is —CH₂—. In yet a furtherembodiment, a is 3, b is 0, R is —CH₂— and Q is —C(O)O—. In anotherembodiment, R¹ and R² are methyl, a is 3, b is 0, R is —CH₂— and Q is—C(O)O—.

In another embodiment, X and Y are each, independently —(CH₂)_(n)—wherein n is 4 to 20, e.g., 4 to 18, 4 to 16, or 4 to 12. In oneembodiment, n is 4, 5, 6, 7, 8, 9, or 10. In one exemplary embodiment, Xand Y are —(CH₂)₆—. In another embodiment, X and Y are —(CH₂)₇—. In yetanother embodiment, X and Y are —(CH₂)₉—. In yet another embodiment, Xand Y are —(CH₂)₈—.

In further embodiments, M¹ and M² are each, independently, —OC(O)— or—C(O)O—. For example, in one embodiment, M¹ and M² are each —C(O)O—.

In another embodiment, the cationic lipid is a compound of formula (II),which has a branched alkyl at the alpha position adjacent to thebiodegradable group (between the biodegradable group and the terminus ofthe tail, i.e., Z¹ or Z²):

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof),wherein

R′ is absent, hydrogen, or alkyl (e.g., C₁-C₄ alkyl);

with respect to R¹ and R²,

-   -   (i) R¹ and R² are each, independently, optionally substituted        alkyl, alkenyl, alkynyl, cycloalkylalkyl, heterocycle, or R¹⁰;    -   (ii) R¹ and R², together with the nitrogen atom to which they        are attached, form an optionally substituted heterocylic ring;        or    -   (iii) one of R¹ and R² is optionally substituted alkyl, alkenyl,        alkynyl, cycloalkyl, cycloalkylalkyl, or heterocycle, and the        other forms a 4-10 member heterocyclic ring or heteroaryl (e.g.,        a 6-member ring) with (a) the adjacent nitrogen atom and (b) the        (R)_(a) group adjacent to the nitrogen atom;

each occurrence of R is, independently, —(CR³R⁴)—;

each occurrence of R³ and R⁴ are, independently H, halogen, OH, alkyl,alkoxy, —NH₂, R¹⁰, alkylamino, or dialkylamino (in one preferredembodiment, each occurrence of R³ and R⁴ are, independently H or C₁-C₄alkyl);

each occurrence of R¹⁰ is independently selected from PEG and polymersbased on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol),poly(glycerol), poly(N-vinylpyrrolidone),poly[N-(2-hydroxypropyl)methacrylamide] and poly(amino acid)s, wherein(i) the PEG or polymer is linear or branched, (ii) the PEG or polymer ispolymerized by n subunits, (iii) n is a number-averaged degree ofpolymerization between 10 and 200 units, and (iv) wherein the compoundof formula has at most two R¹⁰ groups (preferably at most one R¹⁰group);

the dashed line to Q is absent or a bond;

when the dashed line to Q is absent then Q is absent or is —O—, —NH—,—S—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)N(R⁴)—, —N(R⁵)C(O)—, —S—S—,—OC(O)O—, —O—N═C(R⁵)—, —C(R⁵)═N—O—, —OC(O)N(R⁵)—, —N(R⁵)C(O)N(R⁵)—,—N(R⁵)C(O)O—, —C(O)S—, —C(S)O— or —C(R⁵)═N—O—C(O)—; or

when the dashed line to Q is a bond then (i) b is 0 and (ii) Q and thetertiary carbon adjacent to it (C*) form a substituted or unsubstituted,mono- or bi-cyclic heterocyclic group having from 5 to 10 ring atoms(e.g., the heteroatoms in the heterocyclic group are selected from O andS, preferably O);

each occurrence of R⁵ is, independently, H or alkyl;

X and Y are each, independently, alkylene (e.g., C₆-C₈ alkylene) oralkenylene, wherein the alkylene or alkenylene group is optionallysubstituted with one or two fluorine atoms at the alpha position to theM¹ or M² group (e.g.,

M¹ and M² are each, independently, a biodegradable group (e.g., —OC(O)—,—C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O—, —S—S—, —C(R⁵)═N—,—N═C(R⁵)—, —C(R⁵)═N—O—, —O—N═C(R⁵)—, —C(O)(NR⁵)—, —N(R⁵)C(O)—,—C(S)(NR⁵)—, —N(R⁵)C(O)—, —N(R⁵)C(O)N(R⁵)—, —OC(O)O—, —OSi(R⁵)₂O—,—C(O)(CR³R⁴)C(O)O—, —OC(O)(CR³R⁴)C(O)—, or

(wherein R¹¹ is a C₂-C₈ alkyl or alkenyl));

each occurrence of R^(z) is, independently, C₁-C₈ alkyl (e.g., methyl,ethyl, isopropyl);

-   -   a is 1, 2, 3, 4, 5 or 6;

b is 0, 1, 2, or 3; and

Z¹ and Z² are each, independently, C₈-C₁₄ alkyl or C₈-C₁₄ alkenyl,wherein (i) the alkenyl group may optionally be substituted with one ortwo fluorine atoms at the alpha position to a double bond which isbetween the double bond and the terminus of Z¹ or Z² (e.g.,

and (ii) the terminus of at least one of Z¹ and Z² is separated from thegroup M¹ or M² by at least 8 carbon atoms.

In another embodiment, X and Y are each, independently —(CH₂)_(n)—wherein n is 4 to 20, e.g., 4 to 18, 4 to 16, or 4 to 12. In oneembodiment, n is 4, 5, 6, 7, 8, 9, or 10. In one exemplary embodiment, Xand Y are —(CH₂)₆—. In another embodiment, X and Y are —(CH₂)₇—. In yetanother embodiment, X and Y are —(CH₂)₉—. In yet another embodiment, Xand Y are —(CH₂)₈—.

The R′R¹R²N—(R)_(a)-Q-(R)_(b)— group can be any of the head groupsdescribed herein, including those shown in Table 1 below, and saltsthereof. In one preferred embodiment, R′R¹R²N—(R)_(a)-Q-(R)_(b)— is(CH₃)₂N—(CH₂)₃—C(O)O—, (CH₃)₂N—(CH₂)₂—NH—C(O)O—,(CH₃)₂N—(CH₂)₂—OC(O)—NH—, or (CH₃)₂N—(CH₂)₃—C(CH₃)═N—O—.

In another embodiment, the cationic lipid is a compound of formula(III), which has a branching point at a position that is 2-6 carbonatoms (i.e., at the beta (β), gamma (γ), delta (δ), epsilon (ε) or zetaposition (ζ)) adjacent to the biodegradable group (between thebiodegradable group and the terminus of the tail, i.e., Z¹ or Z²):

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof),wherein

R′, R¹, R², R, R³, R⁴, R¹⁰, Q, R⁵, M¹, M², R^(z), a, and b are definedas in formula (I);

L¹ and L² are each, independently, C₁-C₅ alkylene or C₂-C₅ alkenylene;

X and Y are each, independently, alkylene (e.g., C₄ to C₂₀ alkylene orC₆-C₈ alkylene) or alkenylene (e.g., C₄ to C₂₀ alkenylene); and

Z¹ and Z² are each, independently, C₈-C₁₄ alkyl or C₈-C₁₄ alkenyl,wherein the alkenyl group may optionally be substituted with one or twofluorine atoms at the alpha position to a double bond which is betweenthe double bond and the terminus of Z¹ or Z² (e.g.,

and with the proviso that the terminus of at least one of Z¹ and Z² isseparated from the group M¹ or M² by at least 8 carbon atoms.

In one embodiment, L¹ and L² are each —CH₂—. In another embodiment, L¹and L² are each —(CH₂)₂—. In one embodiment, L¹ and L² are each—(CH₂)₃—. In yet another embodiment, L¹ and L² are each —(CH₂)₄—. In yetanother embodiment, L¹ and L² are each —(CH₂)₅—. In yet anotherembodiment, L¹ and L² are each —CH₂—CH═CH—. In a preferred embodiment,L¹ and L² are each —CH₂— or —(CH₂)₂.

In one embodiment, X and Y are each, independently —(CH₂)_(n) wherein nis 4 to 20, e.g., 4 to 18, 4 to 16, or 4 to 12. In one embodiment, n is4, 5, 6, 7, 8, 9, or 10. In one exemplary embodiment, X and Y are—(CH₂)₇—. In another exemplary embodiment, X and Y are —(CH₂)₈—. In yetanother exemplary embodiment, X and Y are —(CH₂)₉—.

The R′R¹R²N—(R)_(a)-Q-(R)_(b)— group can be any of the head groupsdescribed herein, including those shown in Table 1 below, and saltsthereof. In one preferred embodiment, R′R¹R²N—(R)_(a)-Q-(R)_(b)— is(CH₃)₂N—(CH₂)₃—C(O)O—, (CH₃)₂N—(CH₂)₂—NH—C(O)O—,(CH₃)₂N—(CH₂)₂—OC(O)—NH—, or (CH₃)₂N—(CH₂)₃—C(CH₃)═N—O—.

In another embodiment, the cationic lipid is a compound of formula(IIIA), which has a branching point at a position that is 2-6 carbonatoms (i.e., at the beta (β), gamma (γ), delta (δ), epsilon (ε) or zetaposition (ζ)) from the biodegradable groups M¹ and M² (i.e., between thebiodegradable group and the terminus of the tail, i.e., Z¹ or Z²):

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof),wherein

R′, R¹, R², R, R³, R⁴, R¹⁰, Q, R⁵, M¹, M², a, and b are defined as informula (I);

each R^(z) is, independently, C₁-C₈ alkyl (e.g., C₃-C₆ alkyl or C₂-C₃alkyl);

L¹ and L² are each, independently, C₁-C₅ alkylene (e.g., C₂-C₃ alkylene)or C₂-C₅ alkenylene;

X and Y are each, independently, alkylene (e.g., C₄ to C₂₀ alkylene orC₇-C₉ alkylene) or alkenylene (e.g., C₄ to C₂₀ alkenylene or C₇-C₉alkenylene); and

Z¹ and Z² are each, independently, C₁-C₈ alkyl (e.g., C₁-C₆ alkyl, suchas C₁, C₃ or C₅ alkyl) or C₂-C₈ alkenyl (such as C₂-C₆ alkenyl);

wherein said cationic lipid is not one selected from:

In one embodiment, L¹ and L² are each —(CH₂)₂—. In another embodiment,L¹ and L² are each —(CH₂)₃—.

In one embodiment, X and Y are each, independently —(CH₂)_(n) wherein nis 4 to 20, e.g., 4 to 18, 4 to 16, 4 to 12 or 7-9. In one embodiment, nis 4, 5, 6, 7, 8, 9, or 10. In one exemplary embodiment, X and Y are—(CH₂)₇—. In yet another exemplary embodiment, X and Y are —(CH₂)₉.

In one preferred embodiment, M¹ and M² are —C(O)O— (where the carbonylgroup in M¹ and M² is bound to the variable X, and the oxygen atom in M¹and M² is bound to the variable L¹ and L²).

The R′R¹R²N—(R)_(a)-Q-(R)_(b)— group can be any of the head groupsdescribed herein, including those shown in Table 1 below, and saltsthereof. In one preferred embodiment, R′R¹R²N—(R)_(a)-Q-(R)_(b)— is(CH₃)₂N—(CH₂)₃—C(O)O—, (CH₃)₂N—(CH₂)₂—NH—C(O)O—,(CH₃)₂N—(CH₂)₂—OC(O)—NH—, or (CH₃)₂N—(CH₂)₃—C(CH₃)═N—O—.

In one preferred embodiment, Z¹ and Z² are branched alkyl or branchedalkenyl groups.

In one embodiment of formula (IIIA), Z¹, Z², and each R^(z) are C₃-C₈alkyl (such as a C₃-C₆ alkyl). In another embodiment of formula (IIIA),Z¹, Z², and each R^(z) are C₃-C₈ branched alkyl (such as a C₃-C₆branched alkyl). In yet another embodiment of formula (IIIA), Z¹, Z²,and each R^(z) are C₃-C₈ straight alkyl (such as a C₃-C₆ straightalkyl).

In one embodiment of formula (IIIA), the branching point is at thesecond position (the β-position) from the biodegradable groups M¹ and M²in each tail. Z¹, Z², and each R^(z) can be C₃-C₈ alkyl (e.g., a C₃-C₆alkyl), such as a C₃-C₈ branched alkyl (e.g., a C₃-C₆ branched alkyl) ora C₃-C₈ straight alkyl (e.g., a C₃-C₆ straight alkyl). In one preferredembodiment, M¹ and M² are —C(O)O— (where the carbonyl group in M¹ and M²is bound to the variable X, and the oxygen atom in M¹ and M² is bound tothe variable L¹ and/or L²).

In one embodiment of formula (IIIA), the branching point is at the thirdposition (the γ-position) from the biodegradable groups M¹ and M² ineach tail. Z¹, Z², and each R^(z) can be C₃-C₈ alkyl (e.g., a C₃-C₆alkyl), such as a C₃-C₈ branched alkyl (e.g., a C₃-C₆ branched alkyl) ora C₃-C₈ straight alkyl (e.g., a C₃-C₆ straight alkyl). In one preferredembodiment, M¹ and M² are —C(O)O— (where the carbonyl group in M¹ and M²is bound to the variable X, and the oxygen atom in M¹ and M² is bound tothe variable L¹ and/or L²).

In one embodiment of formula (IIIA), the branching point is at the thirdposition (the γ-position) from the biodegradable groups M¹ and M² ineach tail.

In another embodiment of formula (IIIA), M¹ and/or M² are not —O(C(O)—(where the oxygen atom in M¹ and/or M² is bound to the variable X, andthe carbonyl in M¹ and/or M² is bound to the variable L¹ and/or L²). Inyet another embodiment of formula (IIIA), Z¹, Z², and R^(z) are notC₃-C₁₀ cycloalkyl(C₁-C₆ alkyl).

In another embodiment, the cationic lipid is a compound of formula (IV),which has a branching point at a position that is 2-6 carbon atoms(i.e., at beta (β), gamma (γ), delta (δ), epsilon (ε) or zeta position(ζ)) adjacent to the biodegradable group (between the biodegradablegroup and the terminus of the tail, i.e., Z¹ or Z²):

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof),wherein

R′, R¹, R², R, R³, R⁴, R¹⁰, Q, R⁵, M¹, M², R^(z), a, and b are definedas in formula (I);

L¹ and L² and are each, independently, C₁-C₅ alkylene or C₂-C₅alkenylene;

X and Y are each, independently, alkylene or alkenylene (e.g., C₁₂-C₂₀alkylene or C₁₂-C₂₀ alkenylene); and

each occurrence of Z is independently C₁-C₄ alkyl (preferably, methyl).

For example, in one embodiment, -L¹-C(Z)₃ is —CH₂C(CH₃)₃. In anotherembodiment, -L¹-C(Z)₃ is —CH₂CH₂C(CH₃)₃.

In one embodiment, the total carbon atom content of each tail (e.g.,—X-M¹-L¹-C(Z)₃ or —Y-M²-L²-C(Z)₃) is from about 17 to about 26. Forexample, the total carbon atom content can be from about 19 to about 26or from about 21 to about 26.

In another embodiment, X and Y are each, independently —(CH₂)_(n)—wherein n is 4 to 20, e.g., 4 to 18, 4 to 16, or 4 to 12. In oneembodiment, n is 4, 5, 6, 7, 8, 9, or 10. In one exemplary embodiment, Xand Y are —(CH₂)₆—. In another embodiment, X and Y are —(CH₂)₇—. In yetanother embodiment, X and Y are —(CH₂)₉—. In yet another embodiment, Xand Y are —(CH₂)₈—.

In one embodiment, the cationic lipid is a compound of formula (V),which has an alkoxy or thioalkoxy (i.e., —S-alkyl) group substitution onat least one tail:

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof),wherein

R′, R¹, R², R, R³, R⁴, R¹⁰, Q, R⁵, M¹, M², a, and b are defined as informula (I);

X and Y are each, independently, alkylene (e.g., C₆-C₈ alkylene) oralkenylene, wherein the alkylene or alkenylene group is optionallysubstituted with one or two fluorine atoms at the alpha position to theM¹ or M² group (e.g.,

Z¹ and Z² are each, independently, C₈-C₁₄ alkyl or C₈-C₁₄ alkenyl,wherein (i) the C₈-C₁₄ alkyl or C₈-C₁₄ alkenyl of at least one of Z¹ andZ² is substituted by one or more alkoxy (e.g., a C₁-C₄ alkoxy such as—OCH₃) or thioalkoxy (e.g., a C₁-C₄ thioalkoxy such as —SCH₃) groups,and (ii) the alkenyl group may optionally be substituted with one or twofluorine atoms at the alpha position to a double bond which is betweenthe double bond and the terminus of Z¹ or Z² (e.g.,

In one embodiment, the alkoxy substitution on Z¹ and/or Z² is at thebeta position from the M¹ and/or M² group.

In another embodiment, X and Y are each, independently —(CH₂)_(n)—wherein n is 4 to 20, e.g., 4 to 18, 4 to 16, or 4 to 12. In oneembodiment, n is 4, 5, 6, 7, 8, 9, or 10. In one exemplary embodiment, Xand Y are —(CH₂)₆—. In another embodiment, X and Y are —(CH₂)₇—. In yetanother embodiment, X and Y are —(CH₂)₉—. In yet another embodiment, Xand Y are —(CH₂)₈—.

The R′R¹R²N—(R)_(a)-Q-(R)_(b)— group can be any of the head groupsdescribed herein, including those shown in Table 1 below, and saltsthereof. In one preferred embodiment, R′R¹R²N—(R)_(a)-Q-(R)_(b)— is(CH₃)₂N—(CH₂)₃—C(O)O—, (CH₃)₂N—(CH₂)₂—NH—C(O)O—,(CH₃)₂N—(CH₂)₂—OC(O)—NH—, or (CH₃)₂N—(CH₂)₃—C(CH₃)═N—O—.

In one embodiment, the cationic lipid is a compound of formula (VIA),which has one or more fluoro substituents on at least one tail at aposition that is either alpha to a double bond or alpha to abiodegradable group:

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof),wherein

R¹, R², R, a, and b are as defined with respect to formula (I);

Q is absent or is —O—, —NH—, —S—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)N(R⁴)—,—N(R⁵)C(O)—, —S—S—, —OC(O)O—, —O—N═C(R⁵)—, —C(R⁵)═N—O—, —OC(O)N(R⁵)—,—N(R⁵)C(O)N(R⁵)—, —N(R⁵)C(O)O—, —C(O)S—, —C(S)O— or —C(R⁵)═N—O—C(O)—;

R′ is absent, hydrogen, or alkyl (e.g., C₁-C₄ alkyl); and

each of R⁹ and R¹⁰ are independently C₁₂-C₂₄ alkyl (e.g., C₁₂-C₂₀alkyl), C₁₂-C₂₄ alkenyl (e.g., C₁₂-C₂₀ alkenyl), or C₁₂-C₂₄ alkoxy(e.g., C₁₂-C₂₀ alkoxy) (a) having one or more biodegradable groups and(b) optionally substituted with one or more fluorine atoms at a positionwhich is (i) alpha to a biodegradable group and between thebiodegradable group and the tertiary carbon atom marked with an asterisk(*), or (ii) alpha to a carbon-carbon double bond and between the doublebond and the terminus of the R⁹ or R¹⁰ group; each biodegradable groupindependently interrupts the C₁₂-C₂₄ alkyl, alkenyl, or alkoxy group oris substituted at the terminus of the C₁₂-C₂₄ alkyl, alkenyl, or alkoxygroup, wherein

-   -   (i) at least one of R⁹ and R¹⁰ contains a fluoro group;

(ii) the compound does not contain the following moiety:

wherein ---- is an optional bond; and

-   -   (iii) the terminus of R⁹ and R¹⁰ is separated from the tertiary        carbon atom marked with an asterisk (*) by a chain of 8 or more        atoms (e.g., 12 or 14 or more atoms).

In one preferred embodiment, the terminus of R⁹ and R¹⁰ is separatedfrom the tertiary carbon atom marked with an asterisk (*) by a chain of18-22 carbon atoms (e.g., 18-20 carbon atoms).

In another embodiment, the terminus of the R⁹ and/or R¹⁰ has the formula—C(O)O—CF₃.

In another embodiment, the cationic lipid is a compound of formula(VIB), which has one or more fluoro substituents on at least one tail ata position that is either alpha to a double bond or alpha to abiodegradable group:

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof),wherein

R′, R¹, R², R, R³, R⁴, R¹⁰, Q, R⁵, M¹, M², a, and b are defined as informula (I);

X and Y are each, independently, alkylene (e.g., C₆-C₈ alkylene) oralkenylene, wherein the alkylene or alkenylene group is optionallysubstituted with one or two fluorine atoms at the alpha position to theM¹ or M² group (e.g.,

and

Z¹ and Z² are each, independently, C₈-C₁₄ alkyl or C₈-C₁₄ alkenyl,wherein said C₈-C₁₄ alkenyl is optionally substituted by one or morefluorine atoms at a position that is alpha to a double bond (e.g.,

wherein at least one of X, Y, Z¹, and Z² contains a fluorine atom.

In one embodiment, at least one of Z¹ and Z² is substituted by twofluoro groups at a position that is either alpha to a double bond oralpha to a biodegradable group. In one embodiment, at least one of Z¹and Z² has a terminal —CF₃ group at a position that is alpha to abiodegradable group (i.e., at least one of Z¹ and Z² terminates with an—C(O)OCF₃ group).

For example, at least one of Z¹ and Z² may include one or more of thefollowing moieties:

In one embodiment, X and Y are each, independently —(CH₂)_(n) wherein nis 4 to 20, e.g., 4 to 18, 4 to 16, or 4 to 12. In one embodiment, n is4, 5, 6, 7, 8, 9, or 10. In one exemplary embodiment, X and Y are—(CH₂)₇—. In another exemplary embodiment, X and Y are —(CH₂)₉—. In yetanother embodiment, X and Y are —(CH₂)₈—.

The R′R¹R²N—(R)_(a)-Q-(R)_(b)— group can be any of the head groupsdescribed herein, including those shown in Table 1 below, and saltsthereof. In one preferred embodiment, R′R¹R²N—(R)_(a)-Q-(R)_(b)— is(CH₃)₂N—(CH₂)₃—C(O)O—, (CH₃)₂N—(CH₂)₂—NH—C(O)O—,(CH₃)₂N—(CH₂)₂—OC(O)—NH—, or (CH₃)₂N—(CH₂)₃—C(CH₃)═N—O—.

In one embodiment, the cationic lipid is a compound of formula (VII),which has an acetal group as a biodegradable group in at least one tail:

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof),wherein

R′, R¹, R², R, R³, R⁴, R¹⁰, Q, R⁵, a, and b are defined as in formula(I);

X and Y are each, independently, alkylene (e.g., C₆-C₈ alkylene) oralkenylene, wherein the alkylene or alkenylene group is optionallysubstituted with one or two fluorine atoms at the alpha position to theM¹ or M² group (e.g.,

M¹ and M² are each, independently, a biodegradable group (e.g., —OC(O)—,—C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O—, —S—S—, —C(R⁵)═N—,—N═C(R⁵)—, —C(R⁵)═N—O—, —O—N═C(R⁵)—, —C(O)(NR⁵)—, —N(R⁵)C(O)—,—C(S)(NR⁵)—, —N(R⁵)C(O)—, —N(R⁵)C(O)N(R⁵)—, —OC(O)O—, —OSi(R⁵)₂O—,—C(O)(CR³R⁴)C(O)O—, —OC(O)(CR³R⁴)C(O)—, or

(wherein R¹¹ is a C₄-C₁₀ alkyl or C₄-C₁₀ alkenyl));

with the proviso that at least one of M¹ and M² is

and

Z¹ and Z² are each, independently, C₄-C₁₄ alkyl or C₄-C₁₄ alkenyl,wherein the alkenyl group may optionally be substituted with one or twofluorine atoms at the alpha position to a double bond which is betweenthe double bond and the terminus of Z¹ or Z² (e.g.,

In one embodiment, each of M¹ and M² is

In another embodiment, X and Y are each, independently —(CH₂)_(n)—wherein n is 4 to 20, e.g., 4 to 18, 4 to 16, or 4 to 12. In oneembodiment, n is 4, 5, 6, 7, 8, 9, or 10. In one exemplary embodiment, Xand Y are —(CH₂)₆—. In another embodiment, X and Y are —(CH₂)₇—. In yetanother embodiment, X and Y are —(CH₂)₉—. In yet another embodiment, Xand Y are —(CH₂)₈—.

The R′R¹R²N—(R)_(a)-Q-(R)_(b)— group can be any of the head groupsdescribed herein, including those shown in Table 1 below, and saltsthereof. In one preferred embodiment, R′R¹R²N—(R)_(a)-Q-(R)_(b)— is(CH₃)₂N—(CH₂)₃—C(O)O—, (CH₃)₂N—(CH₂)₂—NH—C(O)O—,(CH₃)₂N—(CH₂)₂—OC(O)—NH—, or (CH₃)₂N—(CH₂)₃—C(CH₃)═N—O—.

In another embodiment, the present invention relates to a cationic lipidor a salt thereof having:

(i) a central carbon atom,

(ii) a nitrogen containing head group directly bound to the centralcarbon atom, and

(iii) two hydrophobic tails directly bound to the central carbon atom,wherein each hydrophobic tail is of the formula —R^(e)-M-R^(f) whereR^(e) is a C₄-C₁₄ alkyl or alkenyl, M is a biodegradable group, andR^(f) is a branched alkyl or alkenyl (e.g., a C₁₀-C₂₀ alkyl or C₁₀-C₂₀alkenyl), such that (i) the chain length of —R^(e)-M-R^(f) is at most 20atoms (i.e. the total length of the tail from the first carbon atomafter the central carbon atom to a terminus of the tail is at most 20),and (ii) the group —R^(e)-M-R^(f) has at least 20 carbon atoms (e.g., atleast 21 atoms). Optionally, the alkyl or alkenyl group in R^(e) may besubstituted with one or two fluorine atoms at the alpha position to theM¹ or M² group (e.g.,

Also, optionally, the alkenyl group in R^(f) may be substituted with oneor two fluorine atoms at the alpha position to a double bond which isbetween the double bond and the terminus of R^(f) (e.g.,

In one embodiment, the cationic lipid of the present invention (such asof formulas I-VII) has assymetrical hydrophobic groups (i.e., the twohydrophobic groups have different chemical formulas). For example, thecationic lipid can have the formula:

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof),wherein

G is branched or unbranched C₃-C₁₅ alkyl, alkenyl or alkynyl (e.g., an-C₈ alkyl n-C₉ alkyl, or n-C₁₀ alkyl);

R¹² is a branched or unbranched alkylene or alkenylene (e.g., C₆-C₂₀alkylene or C₆-C₂₀ alkenylene such as C₁₂-C₂₀ alkylene or C₁₂-C₂₀alkenylene);

M₁ is a biodegradable group (e.g., —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—,—OC(S)—, —C(S)O—, —S—S—, —C(R⁵)═N—, —N═C(R⁵)—, —C(R⁵)═N—O—, —O—N═C(R⁵)—,—C(O)(NR⁵)—, —N(R⁵)C(O)—, —C(S)(NR⁵)—, —N(R⁵)C(O)—, —N(R⁵)C(O)N(R⁵)—,—OC(O)O—, —OSi(R⁵)₂O—, —C(O)(CR³R⁴)C(O)O—, —OC(O)(CR³R⁴)C(O)—, or

(wherein R¹¹ is a C₂-C₈ alkyl or alkenyl));

R³ and R⁴ are defined as in formula (I);

each occurrence of R⁵ is, independently, H or alkyl (e.g., C₁-C₄ alkyl);

R¹³ is branched or unbranched C₃-C₁₅ alkyl, alkenyl or alkynyl;

comprises a protonatable group having a pK_(a) of from about 4 to about13, more preferably from about 5 to about 8 (e.g. from about 5 to about7, or from about 5 to about 6.5, or from about 5.5 to about 6.5, or fromabout 6 to about 6.5).

In one embodiment, the primary group includes (i) a head group, and (ii)a central moiety (e.g., a central carbon atom) to which both thehydrophobic tails are directly bonded. Representative central moietiesinclude, but are not limited to, a central carbon atom, a centralnitrogen atom, a central carbocyclic group, a central aryl group, acentral hetrocyclic group (e.g., central tetrahydrofuranyl group orcentral pyrrolidinyl group) and a central heteroaryl group.

Representative

include, but are not limited to,

where n is 0-6.

Representative asymmetrical cationic lipids include:

wherein w is 0, 1, 2, or 3; and x and y are each independently 1, 2, 3,4, 5, 6, or 7.

In a preferred embodiment of the aforementioned biodegradable cationiclipids, the biodegradable cationic lipid has a log P value of at least10.1 (as calculated by the software available athttp://www.molinspiration.com/services/logp.html from MolinspirationCheminformatics of Slovensky Grob, Slovak Republic). More preferably,the log P value is at least 10.2 or 10.3.

In another preferred embodiment of the aforementioned biodegradablecationic lipids, the biodegradable cationic lipid in the lipidnanoparticle has a HPLC retention time (relative to the retention timeof cholesterol in the lipid nanoparticle), hereafter referred to ast_(lipid)−t_(chol), of at least 1.4. (The HPLC parameters are providedin the examples below. Unless otherwise specified, the formulation ofthe lipid nanoparticle used is that described in Example 31). Morepreferably, the t_(lipid)−t_(chol) value is at least 1.75, 2.0, or 2.25.

In another embodiment, the biodegradable cationic lipid of the presentinvention is not one selected from:

In yet another embodiment, the biodegradable cationic lipid is not oneselected from those disclosed in International Publication No. WO2011/153493 and U.S. Patent Publication No. 2012/0027803, both of whichare hereby incorporated by reference.

Yet another embodiment is a biodegradable cationic lipid having (i) alog P value of at least 10.1 and/or a t_(lipid)−t_(chol), of at least1.4, and (2) one or more biodegradable groups (such as an ester group)located in the mid- or distal section of a lipidic moiety (e.g., ahydrophobic chain) of the cationic lipid, with the proviso that thecompound is not selected from

In another embodiment, the biodegradable cationic lipid is not oneselected from those disclosed in International Publication No. WO2011/153493 and U.S. Patent Publication No. 2012/0027803, both of whichare hereby incorporated by reference. The incorporation of thebiodegradable group(s) into the cationic lipid results in fastermetabolism and removal of the cationic lipid from the body followingdelivery of the active pharmaceutical ingredient to a target area. In apreferred embodiment, the cationic lipid includes a branched alkyl orbranched alkenyl group in its biodegradable group(s). In anotherpreferred embodiment, the cationic lipid has a log P of at least 10.2 or10.3. In yet another preferred embodiment, the cationic lipid has at_(lipid)−t_(chol), of at least 1.75, 2.0, or 2.25. The cationic lipidpreferably has a pKa of from about 4 to about 7 (such as 6.0 to 6.5).

In one embodiment, the cationic lipid having a log P value of at least10.1 and/or a t_(lipid)−t_(chol), of at least 1.4 comprises (a) a headgroup (preferably a nitrogen containing head group, such as the headgroups described herein), (b) at least two hydrophobic tails, each ofthe formula -(hydrophobic chain)-(biodegradable group)-(hydrophobicchain), and (c) a linker group (for instance, a single central carbonatom) which is bound to the head group and the hydrophobic tails. Thecationic lipid preferably has one, two, three, four or more of theproperties listed below:

(i) a pKa of from about 4 to about 7 (such as 6.0 to 6.5);

(ii) in at least one hydrophobic tail (and preferably all hydrophobictails), the biodegradable group is separated from the terminus of thehydrophobic tail by from about 6 to about 12 carbon atoms (for instance,6 to 8 carbon atoms or 8 to 12 carbon atoms),

(iii) for at least one hydrophobic tail (and preferably all hydrophobictails), the chain length from the linker group to the terminus of thehydrophobic tail is at most 21 (e.g., at most 20, or from about 17 toabout 21, from about 18 to about 20, or from about 16 to about 18) (Theatom(s) in the linker group are not counted when calculating the chainlength.);

(iv) for at least one hydrophobic tail (and preferably all hydrophobictails), the total number of carbon atoms in the hydrophobic tail is fromabout 17 to about 26 (such as from about 19 to about 26, or from about21 to about 26);

(v) for at least one hydrophobic tail (and preferably all hydrophobictails), the number of carbon atoms between the linker group and thebiodegradable group ranges from about 5 to about 10 (for example, 6 to10, or 7 to 9);

(vi) for at least one hydrophobic tail (and preferably all hydrophobictails), the total number of carbon atoms between the linker group andthe terminus of the hydrophobic tail is from about 15 to about 20 (suchas from 16 to 20, 16 to 18, or 18 to 20);

(vii) for at least one hydrophobic tail (and preferably all hydrophobictails), the total number of carbon atoms between the biodegradable groupand the terminus of the hydrophobic tail is from about 12 to about 18(such as from 13 to 25);

-   -   (viii) for at least one hydrophobic tail (and preferably all        hydrophobic tails), the terminal hydrophobic chain in the        hydrophobic tail is a branched alkyl or alkenyl group, for        example, where the branching occurs at the α, β, γ, or δ        position on the hydrophobic chain relative to the biodegradable        group;

(ix) when formulated as a lipid nanoparticle (such as in Example 35),the cationic lipid has an in vivo half life (t_(1/2)) in the liver ofless than about 3 hours, such as less than about 2.5 hours, less thanabout 2 hours, less than about 1.5 hours, less than about 1 hour, lessthan about 0.5 hour or less than about 0.25 hours;

(x) when formulated as a lipid nanoparticle (such as in Example 35), thecationic lipid is eliminated from the liver in mice with a greater than10-fold reduction in lipid levels relative to C_(max) within the first24 hours post-dose;

(xi) when formulated as a lipid nanoparticle (such as in Example 35),the cationic lipid is eliminated from the spleen in mice with an equalor greater than 10-fold reduction in lipid levels relative to C_(max)within the first 168 hours post-dose; and

-   -   (xii) when formulated as a lipid nanoparticle (such as in        Example 35), the cationic lipid is eliminated from plasma with a        terminal plasma half-life (t½β) in rodents and non-human        primates of 48 hours or shorter.

The present invention embodies compounds having any combination of someor all of the aforementioned properties. These properties provide acationic lipid which remains intact until delivery of an active agent,such as a nucleic acid, after which cleavage of the hydrophobic tailoccurs in vivo. For instance, the compounds can have all of properties(i) to (viii) (in addition to the log P or t_(lipid)−t_(chol) value). Inanother embodiment, the compounds have properties (i), (ii), (iii), and(viii). In yet another embodiment, the compounds have properties (i),(ii), (iii), (v), (vi), and (viii).

Another embodiment is a method of preparing a cationic lipid comprising:

(a) designing a cationic lipid having a log P value of at least 10.1and/or a t_(lipid)−t_(chol), of at least 1.4, and optionally also havingone, two, three, four, or more properties from the list above (i.e.,properties (i)-(xii)); and

-   -   (b) synthesizing the cationic lipid of step (a). The cationic        lipid in step (a) may comprises (a) a head group (preferably a        nitrogen containing head group, such as the head groups        described herein), (b) at least two hydrophobic tails, each of        the formula -(hydrophobic chain)-(biodegradable        group)-(hydrophobic chain), and (c) a linker group (for        instance, a single central carbon atom) which is bound to the        head group and the hydrophobic tails. Step (a) may comprise:

(a)(i) preparing one or more cationic lipids having a log P value of atleast 10.1 and/or a t_(lipid)−t_(chol), of at least 1.4, and optionallyalso having one, two, three, four, or more properties from the listabove (i.e., properties (i)-(xii);

(a)(ii) screening the cationic lipids to determine their efficacy and/ortoxicity in lipid nanoparticles; and

(a)(iii) selecting a cationic lipid for synthesis.

Yet another embodiment is a method of designing a cationic lipidcomprising:

(a) selecting a cationic lipid having a log P value of at least 10.1and/or a t_(lipid)−t_(chol), of at least 1.4, and optionally also havingone, two, three, four, or more properties from the list above (i.e.,properties (i)-(xii)); and

(b) optionally,

-   -   (i) preparing one or more cationic lipids having a log P value        of at least 10.1 and/or a t_(lipid)−t_(chol), of at least 1.4,        and optionally also having one, two, three, four, or more        properties from the list above (i.e., properties (i)-(xii);    -   (ii) screening the cationic lipids to determine their efficacy        and/or toxicity in lipid nanoparticles; and    -   (iii) optionally, selecting a cationic lipid for further        development or use.

In one embodiment, the PEG lipid has the formula:

wherein

G₁ is branched or unbranched C₃-C₁₅ alkyl, alkenyl or alkynyl (e.g., an-C₈ alkyl n-C₉ alkyl, or n-C₁₀ alkyl); or G₁ is —R¹²-M₁-R¹³;

R¹² is a branched or unbranched alkylene or alkenylene (e.g., C₆-C₂₀alkylene or C₆-C₂₀ alkenylene such as C₁₂-C₂₀ alkylene or C₁₂-C₂₀alkenylene);

M₁ is a biodegradable group (e.g., —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—,—OC(S)—, —C(S)O—, —S—S—, —C(R⁵)═N—, —N═C(R⁵)—, —C(R⁵)═N—O—, —O—N═C(R⁵)—,—C(O)(NR⁵)—, —N(R⁵)C(O)—, —C(S)(NR⁵)—, —N(R⁵)C(O)—, —N(R⁵)C(O)N(R⁵)—,—OC(O)O—, —OSi(R⁵)₂O—, —C(O)(CR³R⁴)C(O)O—, —OC(O)(CR³R⁴)C(O)—, or

(wherein R¹¹ is a C₂-C₈ alkyl or alkenyl));

R³ and R⁴ are defined as in formula (I);

-   -   each occurrence of R⁵ is, independently, H or alkyl (e.g., C₁-C₄        alkyl);

R¹³ is branched or unbranched C₃-C₁₅ alkyl, alkenyl or alkynyl;

comprises a PEG moiety, such as

moiety wherein b is an integer from 10 to 1,000 (e.g., 5-100, 10-60,15-50, or 20-45); R³ is —H, —R^(c), or —OR^(c); and R^(c) is —H, alkyl,acyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl.

In one embodiment, the pegylated primary group includes (i) a head grouphaving a PEG moiety, and (ii) a central moiety (e.g., a central carbonatom) to which both the hydrophobic tails are directly bonded.Representative central moieties include, but are not limited to, acentral carbon atom, a central nitrogen atom, a central carbocyclicgroup, a central aryl group, a central hetrocyclic group (e.g., centraltetrahydrofuranyl group or central pyrrolidinyl group) and a centralheteroaryl group.

Representative

include, but are not limited to,

where b is 10-100 (e.g., 20-50 or 40-50)

Another embodiment of the present invention is a PEG lipid (or a saltthereof) having:

(i) a pegylated primary group including a head group which includes aPEG moiety (e.g., having from 10 to 1000 repeating units such as ethoxyunits)), and

(iii) one or more hydrophobic tails (preferably, two hydrophobic tails)directly bound to the pegylated primary group, wherein at least onehydrophobic tail is of the formula —R^(e)-M-R^(f) where R^(e) is aC₄-C₁₄ alkyl or alkenyl, M is a biodegradable group, and R^(f) is abranched alkyl or alkenyl (e.g., a C₁₀-C₂₀ alkyl or C₁₀-C₂₀ alkenyl),such that (i) the chain length of —R^(e)-M-R^(f) is at most 20 atoms(i.e. the total length of the tail from the first carbon atom after thecentral carbon atom to a terminus of the tail is at most 20), and (ii)the group —R^(e)-M-R^(f) has at least 20 carbon atoms (e.g., at least 21atoms). Optionally, the alkyl or alkenyl group in R^(e) may besubstituted with one or two fluorine atoms at the alpha position to theM¹ or M² group (e.g.,

). Also, optionally, the alkenyl group in R^(f) may be substituted withone or two fluorine atoms at the alpha position to a double bond whichis between the double bond and the terminus of R^(f) (e.g.,

In one embodiment, the pegylated primary group includes (i) a head grouphaving a PEG moiety, and (ii) a central moiety (e.g., a central carbonatom) to which the hydrophobic tails are directly bound. The PEG moietymay have 5-100, 10-60, 15-50, or 20-45 repeating units. For example, thePEG moiety may have the formula

moiety wherein b is an integer from 10 to 1,000 (e.g., 5-100, 10-60,15-50, or 20-45); R³ is —H, —R^(c), or —OR^(c); and R^(c) is —H, alkyl(e.g., C₁-C₄ alkyl), acyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, or heterocyclyl.

Yet another embodiment is a lipid particle that includes a cationiclipid and/or PEG lipid of the present invention. In one embodiment, thelipid particle includes a cationic lipid of the present invention (e.g.,of one of formulas (I)-(VIII)). In another embodiment, the lipidparticle includes a PEG lipid of the present invention (e.g., of formula(IX)). In yet another embodiment, the lipid particle includes a cationiclipid of the present invention and a PEG lipid of the present invention.

In a preferred embodiment, the lipid particle includes a neutral lipid,a lipid capable of reducing aggregation, a cationic lipid, andoptionally, a sterol (e.g., cholesterol). Suitable neutral lipidsinclude, but are not limited to, distearoylphosphatidylcholine (DSPC),dipalmitoylphosphatidylcholine (DPPC), POPC, DOPE, and SM. Suitablelipids capable of reducing aggregation include, but are not limited to,a PEG lipid, such as PEG-DMA, PEG-DMG, and those of the presentinvention (e.g., of formula (IX)) or a combination thereof.

The lipid particle may further include an active agent (e.g., atherapeutic agent). The active agent can be a nucleic acid such as aplasmid, an immunostimulatory oligonucleotide, an siRNA, an antisenseoligonucleotide, a microRNA, an antagomir, an aptamer, or a ribozyme. Ina preferred embodiment, the nucleic acid is a siRNA. In anotherpreferred embodiment, the nucleic acid is a miRNA.

In another embodiment, the lipid particle includes a cationic lipid ofthe present invention, a neutral lipid and a sterol. The lipid particlemay further include an active agent, such as a nucleic acid (e.g., ansiRNA or miRNA).

In yet another embodiment, the lipid particle includes a PEG lipid ofthe present invention, a cationic lipid, a neutral lipid, and a sterol.The lipid particle may further include an active agent, such as anucleic acid (e.g., an siRNA or miRNA).

The lipid particles described herein may be lipid nanoparticles.

Yet another embodiment of the invention is a pharmaceutical compositionwhich includes a lipid particle of the present invention and apharmaceutically acceptable carrier.

In one embodiment, the cationic lipid remains intact until delivery ofthe nucleic acid molecule after which cleavage of the hydrophobic tailoccurs in vivo.

In another embodiment, the PEG lipid remains intact until delivery ofthe nucleic acid molecule after which cleavage of the hydrophobic tailoccurs in vivo.

In another embodiment, the present invention relates to a method ofdelivering a nucleic acid molecule comprising administering a nucleiclipid particle comprising (i) the nucleic acid molecule and (ii) acationic lipid and/or a PEG lipid of the present invention. In oneembodiment, the cationic lipid and/or a PEG lipid remains intact untildelivery of the nucleic acid molecule after which cleavage of thehydrophobic tail occurs in vivo.

Yet another aspect is a method of modulating the expression of a targetgene in a cell by providing to the cell a lipid particle of the presentinvention. The active agent can be a nucleic acid selected from aplasmid, an immunostimulatory oligonucleotide, an siRNA, an antisenseoligonucleotide, a microRNA, an antagomir, an aptamer, and a ribozyme.In a preferred embodiment, the nucleic acid is a siRNA or miRNA.

Yet another aspect is a method of treating a disease or disordercharacterized by the overexpression of a polypeptide in a subject byproviding to the subject a pharmaceutical composition of the presentinvention, wherein the active agent is a nucleic acid selected from ansiRNA, a microRNA, and an antisense oligonucleotide, and wherein thesiRNA, microRNA, or antisense oligonucleotide includes a polynucleotidethat specifically binds to a polynucleotide that encodes thepolypeptide, or a complement thereof. In a preferred embodiment, thenucleic acid is a siRNA or miRNA.

Yet another aspect is a method of treating a disease or disordercharacterized by underexpression of a polypeptide in a subject byproviding to the subject a pharmaceutical composition of the presentinvention, wherein the active agent is a plasmid that encodes thepolypeptide or a functional variant or fragment thereof.

Yet another aspect is a method of inducing an immune response in asubject by providing to the subject a pharmaceutical composition whereinthe active agent is an immunostimulatory oligonucleotide.

Yet another aspect is a transfection agent that includes the compositionor lipid particles described above, where the composition or lipidparticles include a nucleic acid. The agent, when contacted with cells,can efficiently deliver nucleic acids to the cells. Yet another aspectis a method of delivering a nucleic acid to the interior of a cell, byobtaining or forming a composition or lipid particles described above,and contacting the composition or lipid particles with a cell.

DETAILED DESCRIPTION

In one aspect, the present invention relates to a lipid particle thatincludes a neutral lipid, a lipid capable of reducing aggregation (e.g.,a PEG lipid), a cationic lipid, and optionally a sterol. In certainembodiments, the lipid particle further includes an active agent (e.g.,a therapeutic agent). Various exemplary embodiments of these lipids,lipid particles and compositions comprising the same, and their use todeliver therapeutic agents and modulate gene and protein expression aredescribed in further detail below.

The Cationic Lipid

In one embodiment, the cationic lipid is a compound of any one ofFormulas I-VIII. The following disclosure represents various embodimentsof the compounds described above, including the compounds of FormulasI-VIII.

In one embodiment, M¹ and M² are each, independently:

—OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O—, —S—S—, —C(R⁵)═N—,—N═C(R⁵)—, —C(R⁵)═N—O—, —O—N═C(R⁵)—, —C(O)(NR⁵)—, —N(R⁵)C(O)—,—C(S)(NR⁵)—, —N(R⁵)C(O)—, —N(R⁵)C(O)N(R⁵)—, —OC(O)O—, —OSi(R⁵)₂O—,—C(O)(CR³R⁴)C(O)O—, —OC(O)(CR³R⁴)C(O)—, or

(wherein R¹¹ is a C₂-C₈ alkyl or alkenyl).

In another embodiment, M¹ and M² are each, independently:

—OC(O)—, —C(O)—O—, —C(R⁵)═N—, —N═C(R⁵)—, —C(R⁵)═N—O—, —O—N═C(R⁵)—,—O—C(O)O—, —C(O)N(R⁵)—, —N(R⁵)C(O)—, —C(O)S—, —SC(O)—, —C(S)O—, —OC(S)—,—OSi(R⁵)₂O—, —C(O)(CR³R⁴)C(O)O—, or —OC(O)(CR³R⁴)C(O)—.

In yet another embodiment, M¹ and M² are each, independently:

—C(O)—O—, —OC(O)—, —C(R⁵)═N—, —C(R⁵)═N—O—, —O—C(O)O—, —C(O)N(R⁵)—,—C(O)S—, —C(S)O—, —OSi(R⁵)₂O—, —C(O)(CR³R⁴)C(O)O—, or—OC(O)(CR³R⁴)C(O)—.

In another embodiment, M¹ and M² are each —C(O)O—.

In one embodiment, R¹ and R² are each, individually, optionallysubstituted alkyl, cycloalkyl, cycloalkylalkyl, or heterocycle. In oneembodiment, R¹ is alkyl and R² is alkyl, cycloalkyl or cycloalkylalkyl.In one embodiment, R¹ and R² are each, individually, alkyl (e.g., C₁-C₄alkyl, such as methyl, ethyl, or isopropyl). In one embodiment, R¹ andR² are both methyl. In another embodiment, R¹ and R², together with thenitrogen atom to which they are attached, form an optionally substitutedheterocylic ring (e.g., N-methylpiperazinyl). In another embodiment, oneof R¹ and R² is

(e.g., R¹ is one of the two aforementioned groups and R² is hydrogen).

In one embodiment, R′ is hydrogen or alkyl. In another embodiment, R′ ishydrogen or methyl. In one embodiment, R′ is absent. In one embodiment,R′ is absent or methyl.

For cationic lipid compounds which contain an atom (e.g., a nitrogenatom) that carries a positive charge, the compound also contains anegatively charged counter ion. The counterion can be any anion, such asan organic or inorganic anion. Suitable examples of anions include, butare not limited to, tosylate, methanesulfonate, acetate, citrate,malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate,α-glycerophosphate, halide (e.g., chloride), sulfate, nitrate,bicarbonate, and carbonate. In one embodiment, the counterion is ahalide (e.g., Cl).

In one embodiment each R is, independently, —(CR³R⁴)—, wherein R³ and R⁴are each, independently, H or alkyl (e.g., C₁-C₄ alkyl). For example, inone embodiment each R is, independently, —(CHR⁴)—, wherein each R⁴ is,independently H or alkyl (e.g., C₁-C₄ alkyl). In another embodiment,each R is, independently, —CH₂—, —C(CH₃)₂— or —CH(iPr)- (where iPr isisopropyl). In another embodiment, each R is —CH₂—.

In another embodiment R⁵ is, in each case, hydrogen or methyl. Forexample, R⁵ can be, in each case, hydrogen.

In one embodiment, Q is absent, —C(O)O—, —OC(O)—, —C(O)N(R⁵)—,—N(R⁵)C(O)—, —S—S—, —OC(O)O—, —C(R⁵)═N—O—, —OC(O)N(R⁵)—,—N(R⁵)C(O)N(R⁵)—, —N(R⁵)C(O)O—, —C(O)S—, —C(S)O— or —C(R⁵)═N—O—C(O)—. Inone embodiment, Q is —C(O)O—.

In one embodiment, the dashed line to Q is absent, b is 0 andR′R¹R²N—(R)_(a)-Q- and the tertiary carbon adjacent to it (C*) form thefollowing group:

where n is 1 to 4 (e.g., n is 2).

In one embodiment, the dashed line to Q is absent, b is 0 andR′R¹R²N—(R)_(a)-Q- and the tertiary carbon adjacent to it form thefollowing group:

where n is 1 to 4 (e.g., n is 2), and R¹, R², R, a, and b are as definedwith respect to formula (I). In one embodiment, a is 3.

In one embodiment, the dashed line to Q is absent, b is 0 andR′R¹R²N—(R)_(a)-Q- and the tertiary carbon adjacent to it form thefollowing group:

where n is 1 to 4 (e.g., n is 2), and R¹, R², R, a, and b are as definedwith respect to formula (I). In one embodiment, a is 0. For example, thegroup can be:

In one embodiment, b is 0. In another embodiment, a is 2, 3, or 4 and bis 0. For example, in one embodiment, a is 3 and b is 0. In anotherembodiment, a is 3, b is 0, and Q is —C(O)O—.

In certain embodiments, the biodegradable group present in the cationiclipid is selected from an ester (e.g., —C(O)O— or —OC(O)—), disulfide(—S—S—), oxime (e.g., —C(H)═N—O— or —O—N═C(H)—), —C(O)—O—, —OC(O)—,—C(R⁵)═N—, —N═C(R⁵)—, —C(R⁵)═N—O—, —O—N═C(R⁵)—, —O—C(O)O—, —C(O)N(R⁵),—N(R⁵)C(O)—, —C(S)(NR⁵)—, (NR⁵)C(S)—, —N(R⁵)C(O)N(R⁵)—, —C(O)S—,—SC(O)—, —C(S)O—, —OC(S)—, —OSi(R⁵)₂O—, —C(O)(CR³R⁴)C(O)O—, or—OC(O)(CR³R⁴)C(O)—.

A suitable cholesterol moiety for the cationic lipids of the presentinvention (including compounds of formulas I-VI) has the formula:

Additional embodiments include a cationic lipid having a head group, oneor more hydrophobic tails, and a central moiety between the head groupand the one or more tails. The head group can include an amine; forexample an amine having a desired pK_(a). The pK_(a) can be influencedby the structure of the lipid, particularly the nature of head group;e.g., the presence, absence, and location of functional groups such asanionic functional groups, hydrogen bond donor functional groups,hydrogen bond acceptor groups, hydrophobic groups (e.g., aliphaticgroups), hydrophilic groups (e.g., hydroxyl or methoxy), or aryl groups.The head group amine can be a cationic amine; a primary, secondary, ortertiary amine; the head group can include one amine group (monoamine),two amine groups (diamine), three amine groups (triamine), or a largernumber of amine groups, as in an oligoamine or polyamine. The head groupcan include a functional group that is less strongly basic than anamine, such as, for example, an imidazole, a pyridine, or a guanidiniumgroup. The head group can be zwitterionic. Other head groups aresuitable as well.

Representative central moieties include, but are not limited to, acentral carbon atom, a central nitrogen atom, a central carbocyclicgroup, a central aryl group, a central hetrocyclic group (e.g., centraltetrahydrofuranyl group or central pyrrolidinyl group) and a centralheteroaryl group. Additionally, the central moiety can include, forexample, a glyceride linker, an acyclic glyceride analog linker, or acyclic linker (including a spiro linker, a bicyclic linker, and apolycyclic linker). The central moiety can include functional groupssuch as an ether, an ester, a phosphate, a phosphonate, aphosphorothioate, a sulfonate, a disulfide, an acetal, a ketal, animine, a hydrazone, or an oxime. Other central moieties and functionalgroups are suitable as well.

In one embodiment, the cationic lipid is a racemic mixture. In anotherembodiment, the cationic lipid is enriched in one diastereomer, e.g. thecationic lipid has at least 95%, at least 90%, at least 80% or at least70% diastereomeric excess. In yet another embodiment, the cationic lipidis enriched in one enantiomer, e.g. the lipid has at least 95%, at least90%, at least 80% or at least 70% enantiomer excess. In yet anotherembodiment, the cationic lipid is chirally pure, e.g. is a singleoptical isomer. In yet another embodiment, the cationic lipid isenriched for one optical isomer.

Where a double bond is present (e.g., a carbon-carbon double bond orcarbon-nitrogen double bond), there can be isomerism in theconfiguration about the double bond (i.e. cis/trans or E/Z isomerism).Where the configuration of a double bond is illustrated in a chemicalstructure, it is understood that the corresponding isomer can also bepresent. The amount of isomer present can vary, depending on therelative stabilities of the isomers and the energy required to convertbetween the isomers. Accordingly, some double bonds are, for practicalpurposes, present in only a single configuration, whereas others (e.g.,where the relative stabilities are similar and the energy of conversionlow) may be present as inseparable equilibrium mixture ofconfigurations.

In some cases, a double-bonded unsaturation is replaced by a cyclicunsaturation. The cyclic unsaturation can be a cycloaliphaticunsaturation, e.g., a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, or cyclooctyl group. In some cases, the cyclic group can bea polycyclic group, e.g., a bicyclic group or tricyclic group. Abicyclic group can be bridged, fused, or have a spiro structure. In somecases, a double bond moiety can be replaced by a cyclopropyl moiety,e.g.,

can be replaced by

The cationic lipid includes one or more biodegradable groups. Thebiodegradable group(s) include one or more bonds that may undergo bondbreaking reactions in a biological environment, e.g., in an organism,organ, tissue, cell, or organelle. Functional groups that contain abiodegradable bond include, for example, esters, dithiols, and oximes.Biodegradation can be a factor that influences the clearance of thecompound from the body when administered to a subject. Biodegredationcan be measured in a cell based assay, where a formulation including acationic lipid is exposed to cells, and samples are taken at varioustime points. The lipid fractions can be extracted from the cells andseparated and analyzed by LC-MS. From the LC-MS data, rates ofbiodegradation (e.g., as t_(1/2) values) can be measured.

For example, the compound

includes an ester linkage in each aliphatic chain, which can undergohydrolysis in a biological environment, for example, when exposed to,e.g., a lipase or an esterase. The structure of the compound, of course,influences the rate at which the compound undergoes biodegradation.Thus, a compound where the methyl substituent is on the other side ofthe biodegradable group such as

would be expected to exhibit a different rate of biodegradation. Greatereffects on that rate would be expected from changes in the structure ofthe compound at the site of hydrolysis. One modification that caninfluence the rate of hydrolysis, and thereby influence the rate ofbiodegradation and clearance from a subject's body, is to make theleaving group of the hydrolysis reaction have a secondary, rather thanprimary, alcohol.

For example, without wishing to be bound by theory, Compound 1 shownabove may be metabolized as shown in the scheme below:

In one embodiment, a cationic lipid of any of the embodiments describedherein has an in vivo half life (t_(1/2)) (e.g., in the liver, spleen orplasma) of less than about 3 hours, such as less than about 2.5 hours,less than about 2 hours, less than about 1.5 hours, less than about 1hour, less than about 0.5 hour or less than about 0.25 hours. Thecationic lipid preferably remains intact, or has a half-life sufficientto form a stable lipid nanoparticle which effectively delivers thedesired active pharmaceutical ingredient (e.g., a nucleic acid) to itstarget but thereafter rapidly degrades to minimize any side effects tothe subject. For instance, in mice, the cationic lipid preferably has at_(1/2) in the spleen of from about 1 to about 7 hours.

In another embodiment, a cationic lipid of any of the embodimentsdescribed herein containing a biodegradable group or groups has an invivo half life (t_(1/2)) (e.g., in the liver, spleen or plasma) of lessthan about 10% (e.g., less than about 7.5%, less than about 5%, lessthan about 2.5%) of that for the same cationic lipid without thebiodegradable group or groups.

Some cationic lipids can be conveniently represented as a hydrophobicgroup combined via a central moiety (such as a carbon atom) with aheadgroup. By way of example, the compound:

can be thought of as a combination of a headgroup, a central moiety, andtwo hydrophobic groups as follows:

The present invention includes compounds composed of any combination ofthe head and hydrophobic groups listed below (in combination with acentral moiety (such as a central carbon atom).

Some suitable head groups include those depicted in Table 1A:

TABLE 1A

Suitable primary groups include, but are not limited to, those that area combination of a head group from table 1A with a central carbon atom.Other suitable primary groups include those in table 1B below:

TABLE 1B

Some suitable hydrophobic tail groups include those depicted in Table1C:

TABLE 1C

Other suitable tail groups includes those of the formula —R¹²-M¹-R¹³where R¹² is a C₄-C₁₄ alkyl or C₄-C₁₄ alkenyl, M¹ is a biodegradablegroup as defined above, and R¹³ is a branched alkyl or alkenyl (e.g., aC₁₀-C₂₀ alkyl or C₁₀-C₂₀ alkenyl), such that (i) the chain length of—R¹²-M¹-R¹³ is at most 21 atoms (i.e., the total length of the tail fromthe first carbon after the tertiary carbon (marked with an asterisk) toa terminus of the tail is at most 21), and (ii) the group —R¹²-M¹-R¹³has at least 20 carbon atoms (e.g., at least 21 or 22 carbon atoms).

In one preferred embodiment, the chain length of —R¹²-M¹-R¹³ is at most21 (e.g., at most 20). For example, the chain length can be from about17 to about 24 or from about 18 to about 20.

In one embodiment, the total carbon atom content of each tail(—R¹²-M¹-R¹³) is from about 17 to about 26. For example, the totalcarbon atom content can be from about 19 to about 26 or from about 21 toabout 26.

In one embodiment, the tail has the formula:

where R¹³ is an alkyl or alkenyl group having from about 13 to about 17carbon atoms, and the total carbon length of the tail from the firstcarbon (the leftmost carbon atom above) to a terminus of the tail is atmost 20. Preferably, the tail has from about 22 to about 26 carbonatoms. In one embodiment, the maximum length of R¹³ from its attachmentpoint to the ester group of the compound is 12 carbon atoms (e.g., themaximum length can be 11 carbon atoms). In one preferred embodiment, thebranch in the alkyl or alkenyl group is at the δ-position or later fromthe point of attachment of R¹³ to the ester group. Suitable R¹³ groupsinclude, but are not limited to

For example, the cationic lipid can be

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof),where R¹³ is selected from the groups mentioned above.

Another example is a tail of the formula

where R¹³ is an alkyl or alkenyl group having from about 13 to about 15carbon atoms, and the total carbon length of the tail from the firstcarbon (i.e., the leftmost carbon atom, which is attached to a tertiarycarbon) to a terminus of the tail is at most 20. Preferably, the tailhas from about 24 to about 26 carbon atoms. In one embodiment, themaximum length of R¹³ from its attachment point to the ester group ofthe compound is 10 carbon atoms (e.g., the maximum length can be 9carbon atoms). In one preferred embodiment, the branch in the alkyl oralkenyl group is at the δ-position or later from the point of attachmentof R¹³ to the ester group. Suitable R¹³ groups include, but are notlimited to

For example, the cationic lipid can be

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof),where R¹³ is selected from the groups above.

The R¹³ group may be derived from a natural product, such asdihydrocitgronellol, lavandulol, phytol, or dihydrophytol. In oneembodiment, the R¹³ group in the tails above is a dihydrocitronellolgroup (either as a racemic group or a chirally pure group):

For example, the cationic lipid having a dihydroitronellol group can be

or a salt thereof.

In another embodiment, the R¹³ group in the tails above is a lavandulolgroup or a homolog of it as shown below:

In another embodiment, the R¹³ group in the tails above is a phytol ordihydrophytol group:

For instance, the cationic lipid can be:

A cationic lipid of the formula:

can also be thought of as a combination of a headgroup, a linker moiety,and two parts of the hydrophobic chains as follows:

Various headgrops, linker moieties, and hydrophobic chains I and II arelisted below. The present invention includes compounds composed of anycombination of the head, linker, hydrophobic chain I, and hydrophobicchain II groups listed below.

TABLE 2A Representative headgroups

R = H, alkyl; X = halogen

R = H, alkyl; X = halogen

R = H, alkyl; X = halogen

(where n is 0-5)

n = 0-6

TABLE 2B Representative linker groups

m = 1-5; n = 0-3

m = 0-5; n = 0-3

n = 0-5

m = 0-5; n = 0-3

m = 0-5; n = 0-3

n = 0-3

n = 0-3

m = 1-4; n/o = 0-3 X = O or S

m = 0-5; n = 0-3

m = 0-5; n = 0-3

m = 0-5; n = 0-3

m = 0-5; n = 0-3

m = 0-5; n = 0-3

m = 0-5; n = 0-3

n = 0-5

m = 1-4; n = 0-3 R = COOH, COOMe, COOEt, CN, CONH2 CONHMe

m = 1-4; n/o = 1-3

n = 1-5

n = 0-5 R = H, Me, Et, Pr, allyl

n = 0-5 R = Me, Et, Pr, allyl R1 = Me, Et, Pr, allyl

n = 0-6

n = 0-6

n = 0-6

m = 0-5; n = 0-3

TABLE 2C Representative hydrophobic chain I and/or Ia, and combinationthereof

p = 0-15

p = 0-15, q = 0-15

p = 0-15, q = 0-15

p = 0-15, q = 1-4, r = 0-15

p = 0-15, q = 1-4, r = 0-15

p = 0-15, q = 0-6

p = 0-15

m = 0-4; n = 0-4; R = Me, Et, Pr, iPr, Bu, iBu

n = 1-7

m = 1-4, n = 1-10, p = 0-15, q = 0-15 R = Me, Et, OMe

TABLE 2D Representative biodegradable moieties I and/or Ia andcombinations thereof

n = 0-6

R = H, Me, Et, cyclic alkyl, alicylic, aromatic

X = CH₂, O. S

TABLE 2E Representative hydrophobic chain II and/or IIa and combinationsthereof

n = 0-6; m = 0-16

n = 0-6

n = 0-8

n = 0-8; m = 0-6

n = 0-8 R = OMe, Me, Et, n-Pr, n-Bu

n = 0-8 R = OMe, Me, Et, Pr

n = 0-8 R = OMe, Me, Et, Pr

m = 0-6; n = 0-6; p = 0-6

m = 0-6; n = 0-6; p = 0-6

m = 0-6; n = 0-6; p = 0-6

m = 0-6; n = 0-6; p = 0-6; q = 0-6

Other cationic lipids of the present invention include those in Table 3below. Each asymmetric carbon atom in the compounds below can be eitherchirally pure (R or S) or racemic. These cationic lipids as well asthose in the working examples (such as Examples 36 and 37) are suitablefor forming nucleic acid-lipid particles.

In another aspect, the present invention relates to a method ofpreparing a compound of Formula I-VII. Suitable exemplary syntheticmethods are illustrated in Schemes 1-27 shown in the Examples sectionbelow.

In one embodiment, the cationic lipid of the present invention isselected from the following compounds, and salts thereof (includingpharmaceutically acceptable salts thereof). These cationic lipids aresuitable for forming nucleic acid-lipid particles.

In another embodiment, the cationic lipid of the present invention isselected from the following compounds, and salts thereof (includingpharmaceutically acceptable salts thereof):

In another embodiment, the cationic lipid of the present invention isselected from the following compounds, and salts thereof (includingpharmaceutically acceptable salts thereof):

Additional representative cationic lipids include, but are not limitedto:

Alternatively, for the compounds above having a head of the formula

(where X can be, for example, —C(O)O—), the head can have one methyleneunit between the X group (or other functional group) and nitrogen atom.For example, the head can be:

Cationic lipids include those having alternative fatty acid groups andother dialkylamino groups than those shown, including those in which thealkyl substituents are different (e.g., N-ethyl-N-methylamino-, andN-propyl-N-ethylamino-).

In certain embodiments, the cationic lipids have at least oneprotonatable or deprotonatable group, such that the lipid is positivelycharged at a pH at or below physiological pH (e.g. pH 7.4), and neutralat a second pH, preferably at or above physiological pH. Such lipids arealso referred to as cationic lipids. It will, of course, be understoodthat the addition or removal of protons as a function of pH is anequilibrium process, and that the reference to a charged or a neutrallipid refers to the nature of the predominant species and does notrequire that all of the lipid be present in the charged or neutral form.The lipids can have more than one protonatable or deprotonatable group,or can be zwiterrionic.

In certain embodiments, protonatable lipids (i.e., cationic lipids) havea pK_(a) of the protonatable group in the range of about 4 to about 11.For example, the lipids can have a pK_(a) of about 4 to about 7, e.g.,from about 5 to about 7, such as from about 5.5 to about 6.8, whenincorporated into lipid particles. Such lipids may be cationic at alower pH formulation stage, while particles will be largely (though notcompletely) surface neutralized at physiological pH around pH 7.4.

In particular embodiments, the lipids are charged lipids. As usedherein, the term “charged lipid” includes, but is not limited to, thoselipids having one or two fatty acyl or fatty alkyl chains and aquaternary amino head group. The quaternary amine carries a permanentpositive charge. The head group can optionally include an ionizablegroup, such as a primary, secondary, or tertiary amine that may beprotonated at physiological pH. The presence of the quaternary amine canalter the pKa of the ionizable group relative to the pKa of the group ina structurally similar compound that lacks the quaternary amine (e.g.,the quaternary amine is replaced by a tertiary amine).

Included in the instant invention is the free form of the cationiclipids described herein, as well as pharmaceutically acceptable saltsand stereoisomers thereof. The cationic lipid can be a protonated saltof the amine cationic lipid. The term “free form” refers to the aminecationic lipids in non-salt form. The free form may be regenerated bytreating the salt with a suitable dilute aqueous base solution such asdilute aqueous NaOH, potassium carbonate, ammonia and sodiumbicarbonate.

The pharmaceutically acceptable salts of the instant cationic lipids canbe synthesized from the cationic lipids of this invention which containa basic or acidic moiety by conventional chemical methods. Generally,the salts of the basic cationic lipids are prepared either by ionexchange chromatography or by reacting the free base with stoichiometricamounts or with an excess of the desired salt-forming inorganic ororganic acid in a suitable solvent or various combinations of solvents.Similarly, the salts of the acidic compounds are formed by reactionswith the appropriate inorganic or organic base.

Thus, pharmaceutically acceptable salts of the cationic lipids of thisinvention include non-toxic salts of the cationic lipids of thisinvention as formed by reacting a basic instant cationic lipids with aninorganic or organic acid. For example, non-toxic salts include thosederived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, nitric and the like, as well as saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic,methanesulfonic, ethane disulfonic, oxalic, isethionic, andtrifluoroacetic (TFA).

When the cationic lipids of the present invention are acidic, suitable“pharmaceutically acceptable salts” refers to salts prepared formpharmaceutically acceptable non-toxic bases including inorganic basesand organic bases. Salts derived from inorganic bases include aluminum,ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganicsalts, manganous, potassium, sodium, and zinc. In one embodiment, thebase is selected from ammonium, calcium, magnesium, potassium andsodium. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as arginine, betainecaffeine, choline, N,N¹-dibenzylethylenediamine, diethylamin,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylaminetripropylamine, and tromethamine.

It will also be noted that the cationic lipids of the present inventionmay potentially be internal salts or zwitterions, since underphysiological conditions a deprotonated acidic moiety in the compound,such as a carboxyl group, may be anionic, and this electronic chargemight then be balanced off internally against the cationic charge of aprotonated or alkylated basic moiety, such as a quaternary nitrogenatom.

One or more additional cationic lipids, which carry a net positivecharge at about physiological pH, in addition to those specificallydescribed above, may also be included in the lipid particles andcompositions described herein. Such cationic lipids include, but are notlimited to N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”);N-(2,3-dioleyloxyl)propyl-N,N—N-triethylammonium chloride (“DOTMA”);N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”);1,2-Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.Cl”);3β-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”),N-(1-(2,3-dioleyloxyl)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine(“DOGS”), 1,2-dileoyl-sn-3-phosphoethanolamine (“DOPE”),1,2-dioleoyl-3-dimethylammonium propane (“DODAP”),N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”). Additionally, a number of commercial preparations ofcationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMAand DOPE, available from GIBCO/BRL), and LIPOFECTAMINE (comprising DOSPAand DOPE, available from GIBCO/BRL).

PEG Lipids

Suitable head groups for the PEG lipids include, but are not limited tothose shown in Table 3 below.

TABLE 3

Representative PEG lipids include, but are not limited to:

wherein

n is an integer from 10 to 100 (e.g. 20-50 or 40-50);

s, s′, t and t′ are independently 0, 1, 2, 3, 4, 5, 6 or 7; and m is 1,2, 3, 4, 5, or 6.

Other representative PEG lipids include, but are not limited to:

The Other Lipid Components

The lipid particles and compositions described herein may also includeone or more neutral lipids. Neutral lipids, when present, can be any ofa number of lipid species which exist either in an uncharged or neutralzwitterionic form at physiological pH. Such lipids include, for example,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. In oneembodiment, the neutral lipid component is a lipid having two acylgroups (e.g., diacylphosphatidylcholine anddiacylphosphatidylethanolamine). In one embodiment, the neutral lipidcontains saturated fatty acids with carbon chain lengths in the range ofC₁₀ to C₂₀. In another embodiment, the neutral lipid includes mono ordiunsaturated fatty acids with carbon chain lengths in the range of C₁₀to C₂₀. Suitable neutral lipids include, but are not limited to, DSPC,DPPC, POPC, DOPE, DSPC, and SM.

The lipid particles and compositions described herein may also includeone or more lipids capable of reducing aggregation. Examples of lipidsthat reduce aggregation of particles during formation includepolyethylene glycol (PEG)-modified lipids (PEG lipids, such as PEG-DMGand PEG-DMA), monosialoganglioside Gm1, and polyamide oligomers (“PAO”)such as (described in U.S. Pat. No. 6,320,017, which is incorporated byreference in its entirety). Suitable PEG lipids include, but are notlimited to, PEG-modified phosphatidylethanolamine and phosphatidic acid,PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) (such as thosedescribed in U.S. Pat. No. 5,820,873, incorporated herein by reference),PEG-modified dialkylamines and PEG-modified1,2-diacyloxypropan-3-amines, PEG-modified diacylglycerols anddialkylglycerols, mPEG (mw2000)-diastearoylphosphatidylethanolamine(PEG-DSPE).

The lipid particles and compositions may include a sterol, such ascholesterol.

Lipid Particles

In a further aspect, the present invent relates to lipid particles thatinclude one or more of the cationic lipids described herein. In oneembodiment, the lipid particle includes one or more compounds of formulaI-VII.

Lipid particles include, but are not limited to, liposomes. As usedherein, a liposome is a structure having lipid-containing membranesenclosing an aqueous interior.

Another embodiment is a nucleic acid-lipid particle (e.g., a SNALP)comprising a cationic lipid of the present invention, a non-cationiclipid (such as a neutral lipid), optionally a PEG-lipid conjugate (suchas the lipids for reducing aggregation of lipid particles discussedherein), optionally a sterol (e.g., cholesterol), and a nucleic acid. Asused herein, the term “SNALP” refers to a stable nucleic acid-lipidparticle. A SNALP represents a particle made from lipids, wherein thenucleic acid (e.g., an interfering RNA) is encapsulated within thelipids. In certain instances, SNALPs are useful for systemicapplications, as they can exhibit extended circulation lifetimesfollowing intravenous (i.v.) injection, they can accumulate at distalsites (e.g., sites physically separated from the administration site),and they can mediate silencing of target gene expression at these distalsites. The nucleic acid may be complexed with a condensing agent andencapsulated within a SNALP as set forth in International PublicationNo. WO 00/03683, the disclosure of which is herein incorporated byreference in its entirety.

For example, the lipid particle may include a cationic lipid, afusion-promoting lipid (e.g., DPPC), a neutral lipid, cholesterol, and aPEG-modified lipid. In one embodiment, the lipid particle includes theabove lipid mixture in molar ratios of about 20-70% cationic lipid:0.1-50% fusion promoting lipid: 5-45% neutral lipid: 20-55% cholesterol:0.5-15% PEG-modified lipid (based upon 100% total moles of lipid in thelipid particle).

In another embodiment of the lipid particle, the cationic lipid ispresent in a mole percentage of about 20% and about 60%; the neutrallipid is present in a mole percentage of about 5% to about 25%; thesterol is present in a mole percentage of about 25% to about 55%; andthe PEG lipid is PEG-DMA, PEG-DMG, or a combination thereof, and ispresent in a mole percentage of about 0.5% to about 15% (based upon 100%total moles of lipid in the lipid particle).

In particular embodiments, the molar lipid ratio, with regard to mol %cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA) is approximately40/10/40/10, 35/15/40/10 or 52/13/30/5. This mixture may be furthercombined with a fusion-promoting lipid in a molar ratio of 0.1-50%,0.1-50%, 0.5-50%, 1-50%, 5%-45%, 10%-40%, or 15%-35%. In other words,when a 40/10/40/10 mixture of lipid/DSPC/Chol/PEG-DMG or PEG-DMA iscombined with a fusion-promoting peptide in a molar ratio of 50%, theresulting lipid particles can have a total molar ratio of (mol %cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA/fusion-promoting peptide)20/5/20/5/50. In another embodiment, the neutral lipid, DSPC, in thesecompositions is replaced with POPC, DPPC, DOPE or SM.

In one embodiment, the lipid particles comprise a cationic lipid of thepresent invention, a neutral lipid, a sterol and a PEG-modified lipid.In one embodiment, the lipid particles include from about 25% to about75% on a molar basis of cationic lipid, e.g., from about 35 to about65%, from about 45 to about 65%, about 60%, about 57.5%, about 57.1%,about 50% or about 40% on a molar basis. In one embodiment, the lipidparticles include from about 0% to about 15% on a molar basis of theneutral lipid, e.g., from about 3 to about 12%, from about 5 to about10%, about 15%, about 10%, about 7.5%, about 7.1% or about 0% on a molarbasis. In one embodiment, the neutral lipid is DPPC. In one embodiment,the neutral lipid is DSPC. In one embodiment, the formulation includesfrom about 5% to about 50% on a molar basis of the sterol, e.g., about15 to about 45%, about 20 to about 40%, about 48%, about 40%, about38.5%, about 35%, about 34.4%, about 31.5% or about 31% on a molarbasis. In one embodiment, the sterol is cholesterol.

The lipid particles described herein may further include one or moretherapeutic agents. In a preferred embodiment, the lipid particlesinclude a nucleic acid (e.g., an oligonucleotide), such as siRNA ormiRNA.

In one embodiment, the lipid particles include from about 0.1% to about20% on a molar basis of the PEG-modified lipid, e.g., about 0.5 to about10%, about 0.5 to about 5%, about 10%, about 5%, about 3.5%, about 1.5%,about 0.5%, or about 0.3% on a molar basis. In one embodiment, thePEG-modified lipid is PEG-DMG. In one embodiment, the PEG-modified lipidis PEG-c-DMA. In one embodiment, the lipid particles include 25-75% ofcationic lipid, 0.5-15% of the neutral lipid, 5-50% of the sterol, and0.5-20% of the PEG-modified lipid on a molar basis.

In one embodiment, the lipid particles include 35-65% of cationic lipid,3-12% of the neutral lipid, 15-45% of the sterol, and 0.5-10% of thePEG-modified lipid on a molar basis. In one embodiment, the lipidparticles include 45-65% of cationic lipid, 5-10% of the neutral lipid,25-40% of the sterol, and 0.5-5% of the PEG-modified lipid on a molarbasis. In one embodiment, the PEG modified lipid comprises a PEGmolecule of an average molecular weight of 2,000 Da. In one embodiment,the PEG modified lipid is PEG-distyryl glycerol (PEG-DSG).

In one embodiment, the ratio of lipid:siRNA is at least about 0.5:1, atleast about 1:1, at least about 2:1, at least about 3:1, at least about4:1, at least about 5:1, at least about 6:1, at least about 7:1, atleast about 11:1 or at least about 33:1. In one embodiment, the ratio oflipid:siRNA ratio is between about 1:1 to about 35:1, about 3:1 to about15:1, about 4:1 to about 15:1, or about 5:1 to about 13:1. In oneembodiment, the ratio of lipid:siRNA ratio is between about 0.5:1 toabout 12:1.

In one embodiment, the lipid particles are nanoparticles. In additionalembodiments, the lipid particles have a mean diameter size of from about50 nm to about 300 nm, such as from about 50 nm to about 250 nm, forexample, from about 50 nm to about 200 nm.

In one embodiment, a lipid particle containing a cationic lipid of anyof the embodiments described herein has an in vivo half life (t_(1/2))(e.g., in the liver, spleen or plasma) of less than about 3 hours, suchas less than about 2.5 hours, less than about 2 hours, less than about1.5 hours, less than about 1 hour, less than about 0.5 hour or less thanabout 0.25 hours.

In another embodiment, a lipid particle containing a cationic lipid ofany of the embodiments described herein has an in vivo half life (t₁₁₂)(e.g., in the liver, spleen or plasma) of less than about 10% (e.g.,less than about 7.5%, less than about 5%, less than about 2.5%) of thatfor the same cationic lipid without the biodegradable group or groups.

Additional Components

The lipid particles and compositions described herein can furtherinclude one or more antioxidants. The antioxidant stabilizes the lipidparticle and prevents, decreases, and/or inhibits degradation of thecationic lipid and/or active agent present in the lipid particles. Theantioxidant can be a hydrophilic antioxidant, a lipophilic antioxidant,a metal chelator, a primary antioxidant, a secondary antioxidant, saltsthereof, and mixtures thereof. In certain embodiments, the antioxidantcomprises a metal chelator such as EDTA or salts thereof, alone or incombination with one, two, three, four, five, six, seven, eight, or moreadditional antioxidants such as primary antioxidants, secondaryantioxidants, or other metal chelators. In one preferred embodiment, theantioxidant comprises a metal chelator such as EDTA or salts thereof ina mixture with one or more primary antioxidants and/or secondaryantioxidants. For example, the antioxidant may comprise a mixture ofEDTA or a salt thereof, a primary antioxidant such as a-tocopherol or asalt thereof, and a secondary antioxidant such as ascorbyl palmitate ora salt thereof. In one embodiment, the antioxidant comprises at leastabout 100 mM citrate or a salt thereof. Examples of antioxidantsinclude, but are not limited to, hydrophilic antioxidants, lipophilicantioxidants, and mixtures thereof. Non-limiting examples of hydrophilicantioxidants include chelating agents (e.g., metal chelators) such asethylenediaminetetraacetic acid (EDTA), citrate, ethylene glycoltetraacetic acid (EGTA),1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),diethylene triamine pentaacetic acid (DTPA),2,3-dimercapto-1-propanesulfonic acid (DMPS), dimercaptosuccinic acid(DMSA), cc-lipoic acid, salicylaldehyde isonicotinoyl hydrazone (SIH),hexyl thioethylamine hydrochloride (HTA), desferrioxamine, saltsthereof, and mixtures thereof. Additional hydrophilic antioxidantsinclude ascorbic acid, cysteine, glutathione, dihydrolipoic acid,2-mercaptoethane sulfonic acid, 2-mercaptobenzimidazole sulfonic acid,6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, sodiummetabisulfite, salts thereof, and mixtures thereof. Non-limitingexamples of lipophilic antioxidants include vitamin E isomers such asα-, β-, γ-, and δ-tocopherols and α-, β-, γ-, and δ-tocotrienols;polyphenols such as 2-tert-butyl-4-methyl phenol, 2-tert-butyl-5-methylphenol, and 2-tert-butyl-6-methyl phenol; butylated hydroxyanisole (BHA)(e.g., 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole);butylhydroxytoluene (BHT); tert-butylhydroquinone (TBHQ); ascorbylpalmitate; rc-propyl gallate; salts thereof; and mixtures thereof.Suitable antioxidants and formulations containing such antioxidants aredescribed in International Publication No. WO 2011/066651, which ishereby incorporated by reference.

In another embodiment, the lipid particles or compositions contain theantioxidant EDTA (or a salt thereof), the antioxidant citrate (or a saltthereof), or EDTA (or a salt thereof) in combination with one or more(e.g., a mixture of) primary and/or secondary antioxidants such asα-tocopherol (or a salt thereof) and/or ascorbyl palmitate (or a saltthereof).

In one embodiment, the antioxidant is present in an amount sufficient toprevent, inhibit, or reduce the degradation of the cationic lipidpresent in the lipid particle. For example, the antioxidant may bepresent at a concentration of at least about or about 0.1 mM, 0.5 mM, 1mM, 10 mM, 100 mM, 500 mM, 1 M, 2 M, or 5M, or from about 0.1 mM toabout 1 M, from about 0.1 mM to about 500 mM, from about 0.1 mM to about250 mM, or from about 0.1 mM to about 100 mM.

The lipid particles and compositions described herein can furtherinclude an apolipoprotein. As used herein, the term “apolipoprotein” or“lipoprotein” refers to apolipoproteins known to those of skill in theart and variants and fragments thereof and to apolipoprotein agonists,analogues or fragments thereof described below.

In a preferred embodiment, the active agent is a nucleic acid, such as asiRNA. For example, the active agent can be a nucleic acid encoded witha product of interest, including but not limited to, RNA, antisenseoligonucleotide, an antagomir, a DNA, a plasmid, a ribosomal RNA (rRNA),a micro RNA (miRNA) (e.g., a miRNA which is single stranded and 17-25nucleotides in length), transfer RNA (tRNA), a small interfering RNA(siRNA), small nuclear RNA (snRNA), antigens, fragments thereof,proteins, peptides, vaccines and small molecules or mixtures thereof. Inone more preferred embodiment, the nucleic acid is an oligonucleotide(e.g., 15-50 nucleotides in length (or 15-30 or 20-30 nucleotides inlength)). An siRNA can have, for instance, a duplex region that is 16-30nucleotides long. In another embodiment, the nucleic acid is animmunostimulatory oligonucleotide, decoy oligonucleotide, supermir,miRNA mimic, or miRNA inhibitor. A supermir refers to a single stranded,double stranded or partially double stranded oligomer or polymer ofribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or both ormodifications thereof, which has a nucleotide sequence that issubstantially identical to an miRNA and that is antisense with respectto its target. miRNA mimics represent a class of molecules that can beused to imitate the gene silencing ability of one or more miRNAs. Thus,the term “microRNA mimic” refers to synthetic non-coding RNAs (i.e. themiRNA is not obtained by purification from a source of the endogenousmiRNA) that are capable of entering the RNAi pathway and regulating geneexpression.

The nucleic acid that is present in a lipid-nucleic acid particle can bein any form. The nucleic acid can, for example, be single-stranded DNAor RNA, or double-stranded DNA or RNA, or DNA-RNA hybrids. Non-limitingexamples of double-stranded RNA include siRNA. Single-stranded nucleicacids include, e.g., antisense oligonucleotides, ribozymes, microRNA,and triplex-forming oligonucleotides. The lipid particles of the presentinvention can also deliver nucleic acids which are conjugated to one ormore ligands.

Pharmaceutical Compositions

The lipid particles, particularly when associated with a therapeuticagent, may be formulated as a pharmaceutical composition, e.g., whichfurther comprises a pharmaceutically acceptable diluent, excipient, orcarrier, such as physiological saline or phosphate buffer.

The resulting pharmaceutical preparations may be sterilized byconventional, well known sterilization techniques. The aqueous solutionscan then be packaged for use or filtered under aseptic conditions andlyophilized, the lyophilized preparation being combined with a sterileaqueous solution prior to administration. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, and tonicity adjusting agents, for example, sodium acetate,sodium lactate, sodium chloride, potassium chloride, and calciumchloride. Additionally, the lipidic suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as α-tocopherol and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

The concentration of lipid particle or lipid-nucleic acid particle inthe pharmaceutical formulations can vary, for example, from less thanabout 0.01%, to at or at least about 0.05-5% to as much as 10 to 30% byweight.

Methods of Manufacture

Methods of making cationic lipids, lipid particles containing them, andpharmaceutical compositions containing the cationic lipids and/or lipidparticles are described in, for example, International Publication Nos.WO 2010/054406, WO 2010/054401, WO 2010/054405, WO 2010/054384, WO2010/042877, WO 2010/129709, WO 2009/086558, and WO 2008/042973, andU.S. Patent Publication Nos. 2004/0142025, 2006/0051405 and2007/0042031, each of which is incorporated by reference in itsentirety.

For example, in one embodiment, a solution of one or more lipids(including a cationic lipid of any of the embodiments described herein)in an organic solution (e.g., ethanol) is prepared. Similarly, asolution of one or more active (therapeutic) agents (such as, forexample an siRNA molecule or a 1:1 molar mixture of two siRNA molecules)in an aqueous buffered (e.g., citrate buffer) solution is prepared. Thetwo solutions are mixed and diluted to form a colloidal suspension ofsiRNA lipid particles. In one embodiment, the siRNA lipid particles havean average particle size of about 80-90 nm. In further embodiments, thedispersion may be filtered through 0.45/2 micron filters, concentratedand diafiltered by tangential flow filtration.

DEFINITIONS

As used herein, the term “cationic lipid” inlcudes those lipids havingone or two fatty acid or fatty aliphatic chains and an amino acidcontaining head group that may be protonated to form a cationic lipid atphysiological pH. In some embodiments, a cationic lipid is referred toas an “amino acid conjugate cationic lipid.”

A subject or patient in whom administration of the complex is aneffective therapeutic regimen for a disease or disorder is preferably ahuman, but can be any animal, including a laboratory animal in thecontext of a clinical trial or screening or activity experiment. Thus,as can be readily appreciated by one of ordinary skill in the art, themethods, compounds and compositions of the present invention areparticularly suited to administration to any animal, particularly amammal, and including, but by no means limited to, humans, domesticanimals, such as feline or canine subjects, farm animals, such as butnot limited to bovine, equine, caprine, ovine, and porcine subjects,wild animals (whether in the wild or in a zoological garden), researchanimals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, andcats, avian species, such as chickens, turkeys, and songbirds, i.e., forveterinary medical use.

Many of the chemical groups recited in the generic formulas above arewritten in a particular order (for example, —OC(O)—). It is intendedthat the chemical group is to be incorporated into the generic formulain the order presented unless indicated otherwise. For example, ageneric formula of the form —(R)_(i)-(M¹)_(k)-(R)_(m)— where M¹ is—C(O)O— and k is 1 refers to —(R)_(i)—C(O)O—(R)_(m)— unless specifiedotherwise. It is to be understood that when a chemical group is writtenin a particular order, the reverse order is also contemplated unlessotherwise specified. For example, in a generic formula—(R)_(i)—(M¹)_(k)-(R)_(m)— where M¹ is defined as —C(O)NH— (i.e.,—(R)_(i)—C(O)—NH—(R)_(m)—), the compound where M¹ is —NHC(O)— (i.e.,—(R)_(i)—NHC(O)—(R)_(m)—) is also contemplated unless otherwisespecified.

The term “biodegradable cationic lipid” refers to a cationic lipidhaving one or more biodegradable groups located in the mid- or distalsection of a lipidic moiety (e.g., a hydrophobic chain) of the cationiclipid. The incorporation of the biodegradable group(s) into the cationiclipid results in faster metabolism and removal of the cationic lipidfrom the body following delivery of the active pharmaceutical ingredientto a target area.

As used herein, the term “biodegradable group” refers to a group thatinclude one or more bonds that may undergo bond breaking reactions in abiological environment, e.g., in an organism, organ, tissue, cell, ororganelle. For example, the biodegradable group may be metabolizable bythe body of a mammal, such as a human (e.g., by hydrolysis). Some groupsthat contain a biodegradable bond include, for example, but are notlimited to esters, dithiols, and oximes. Non-limiting examples ofbiodegradable groups are —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—,—C(S)O—, —S—S—, —C(R⁵)═N—, —N═C(R⁵)—, —C(R⁵)═N—O—, —O—N═C(R⁵)—,—C(O)(NR⁵)—, —N(R⁵)C(O)—, —C(S)(NR⁵)—, —N(R⁵)C(O)—, —N(R⁵)C(O)N(R⁵)—,—OC(O)O—, —OSi(R⁵)₂O—, —C(O)(CR³R⁴)C(O)O—, or —OC(O)(CR³R⁴)C(O)—.

As used herein, an “aliphatic” group is a non-aromatic group in whichcarbon atoms are linked into chains, and is either saturated orunsaturated.

The terms “alkyl” and “alkylene” refer to a straight or branched chainsaturated hydrocarbon moiety. In one embodiment, the alkyl group is astraight chain saturated hydrocarbon. Unless otherwise specified, the“alkyl” or “alkylene” group contains from 1 to 24 carbon atoms.Representative saturated straight chain alkyl groups include methyl,ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl. Representativesaturated branched alkyl groups include isopropyl, sec-butyl, isobutyl,tert-butyl, and isopentyl.

The term “alkenyl” refers to a straight or branched chain hydrocarbonmoiety having one or more carbon-carbon double bonds. In one embodiment,the alkenyl group contains 1, 2, or 3 double bonds and is otherwisesaturated. Unless otherwise specified, the “alkenyl” group contains from2 to 24 carbon atoms. Alkenyl groups include both cis and trans isomers.Representative straight chain and branched alkenyl groups includeethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl,2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, and2,3-dimethyl-2-butenyl.

The term “alkynyl” refers to a straight or branched chain hydrocarbonmoiety having one or more carbon-carbon triple bonds. Unless otherwisespecified, the “alkynyl” group contains from 2 to 24 carbon atoms.Representative straight chain and branched alkynyl groups includeacetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, and3-methyl-1-butynyl.

Unless otherwise specified, the terms “branched alkyl”, “branchedalkenyl”, and “branched alkynyl” refer to an alkyl, alkenyl, or alkynylgroup in which one carbon atom in the group (1) is bound to at leastthree other carbon atoms and (2) is not a ring atom of a cyclic group.For example, a spirocyclic group in an alkyl, alkenyl, or alkynyl groupis not considered a point of branching.

Unless otherwise specified, the term “acyl” refers to a carbonyl groupsubstituted with hydrogen, alkyl, partially saturated or fully saturatedcycloalkyl, partially saturated or fully saturated heterocycle, aryl, orheteroaryl. For example, acyl groups include groups such as(C₁-C₂₀)alkanoyl (e.g., formyl, acetyl, propionyl, butyryl, valeryl,caproyl, and t-butylacetyl), (C₃-C₂₀)cycloalkylcarbonyl (e.g.,cyclopropylcarbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl, andcyclohexylcarbonyl), heterocyclic carbonyl (e.g., pyrrolidinylcarbonyl,pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl, piperazinylcarbonyl, andtetrahydrofuranylcarbonyl), aroyl (e.g., benzoyl) and heteroaroyl (e.g.,thiophenyl-2-carbonyl, thiophenyl-3-carbonyl, furanyl-2-carbonyl,furanyl-3-carbonyl, 1H-pyrroyl-2-carbonyl, 1H-pyrroyl-3-carbonyl, andbenzo[b]thiophenyl-2-carbonyl).

The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclichydrocarbon ring system. Unless otherwise specified, the “aryl” groupcontains from 6 to 14 carbon atoms. Examples of aryl moieties include,but are not limited to, phenyl, naphthyl, anthracenyl, and pyrenyl.

The terms “cycloalkyl” and “cycloalkylene” refer to a saturatedmonocyclic or bicyclic hydrocarbon moiety such as cyclopropyl,cyclobutyl, cyclopentyl, and cyclohexyl. Unless otherwise specified, the“cycloalkyl” or “cycloalkylene” group contains from 3 to 10 carbonatoms.

The term “cycloalkylalkyl” refers to a cycloalkyl group bound to analkyl group, where the alkyl group is bound to the rest of the molecule.

The term “heterocycle” (or “heterocyclyl”) refers to a non-aromatic 5-to 8-membered monocyclic, or 7- to 12-membered bicyclic, or 11- to14-membered tricyclic ring system which is either saturated orunsaturated, and which contains from 1 to 3 heteroatoms if monocyclic,1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic,independently selected from nitrogen, oxygen and sulfur, and wherein thenitrogen and sulfur heteroatoms may be optionally oxidized, and thenitrogen heteroatom may be optionally quaternized. For instance, theheterocycle may be a cycloalkoxy group. The heterocycle may be attachedto the rest of the molecule via any heteroatom or carbon atom in theheterocycle. Heterocycles include, but are not limited to, morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl,valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, and tetrahydrothiopyranyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,7-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, where the heteroatoms are selected from O, N,or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or Sif monocyclic, bicyclic, or tricyclic, respectively). The heteroarylgroups herein described may also contain fused rings that share a commoncarbon-carbon bond.

The term “substituted”, unless otherwise indicated, refers to thereplacement of one or more hydrogen radicals in a given structure withthe radical of a specified substituent including, but not limited to:halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio,oxo, thioxy, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl,alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy,aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl,aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro,alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino,hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl,aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonicacid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and analiphatic group. It is understood that the substituent may be furthersubstituted. Exemplary substituents include amino, alkylamino,dialkylamino, and cyclic amino compounds.

The term “halogen” or “halo” refers to fluoro, chloro, bromo and iodo.

The following abbreviations may be used in this application:

DSPC: distearoylphosphatidylcholine; DPPC:1,2-Dipalmitoyl-sn-glycero-3-phosphocholine; POPC:1-palmitoyl-2-oleoyl-sn-phosphatidylcholine; DOPE:1,2-dileoyl-sn-3-phosphoethanolamine; PEG-DMG generally refers to1,2-dimyristoyl-sn-glycerol-methoxy polyethylene glycol (e.g., PEG2000); TBDPSCl: tert-Butylchlorodiphenylsilane; DMAP:dimethylaminopyridine; HMPA: hexamethylphosphoramide; EDC:1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; DIPEA:diisopropylethylamine; DCM: dichloromethane; TEA: triethylamine; TBAF:tetrabutylammonium fluoride

Methods to prepare various organic groups and protective groups areknown in the art and their use and modification is generally within theability of one of skill in the art (see, for example, Green, T. W. et.al., Protective Groups in Organic Synthesis (1999); Stanley R. Sandlerand Wolf Karo, Organic Functional Group Preparations (1989); Greg T.Hermanson, Bioconjugate Techniques (1996); and Leroy G. Wade, CompendiumOf Organic Synthetic Methods (1980)). Briefly, protecting groups are anygroup that reduces or eliminates unwanted reactivity of a functionalgroup. A protecting group can be added to a functional group to mask itsreactivity during certain reactions and then removed to reveal theoriginal functional group. In some embodiments an “alcohol protectinggroup” is used. An “alcohol protecting group” is any group whichdecreases or eliminates unwanted reactivity of an alcohol functionalgroup. Protecting groups can be added and removed using techniques wellknown in the art.

The compounds may be prepared by at least one of the techniquesdescribed herein or known organic synthesis techniques.

EXAMPLES Example 1

Compound 2: To a solution of compound 1 (10.0 g, 18.8 mmol, seeInternational Publication No. WO 2010/054406) in CH₂Cl₂ (80 mL) wereadded triethylamine (7.86 mL, 56.4 mmol), DMAP (459 mg, 3.76 mmol) andtert-butyl(chloro)diphenylsilane (9.62 mL, 37.6 mmol). The reactionmixture was stirred for 24 hours. The mixture was then diluted withCH₂Cl₂ and washed with aqueous saturated NaHCO₃ solution. The organiclayer was separated and dried over anhydrous Na₂SO₄. After filtrationand concentration, the crude product was purified by silica gel columnchromatography (0-5% EtOAc in hexane) to afford 2 (12.4 g, 16.1 mmol,86%, R_(f)=0.24 with hexane). ¹H NMR (400 MHz, CDCl₃) δ 7.66-7.68 (m,4H), 7.33-7.42 (m, 6H), 5.30-5.39 (m, 4H), 3.67-3.72 (m, 1H), 1.97-2.04(m, 8H), 1.07-1.42 (m, 52H), 1.05 (s, 9H), 0.88 (t, J=6.8 Hz, 6H).

Compound 3: To a solution of 2 (12.4 g, 16.1 mmol) in tert-butanol (100mL), THF (30 mL) and H₂O (10 mL) were added 4-methylmorpholine N-oxide(4.15 g, 35.4 mmol) and osmium tetroxide (41 mg, 0.161 mg). The reactionmixture was stirred for 16 hours, then quenched by adding sodiumbisulfite. After removing the solvents by evaporation, the residue wasextracted with Et₂O (500 mL) and H₂O (300 mL). The organic layer wasseparated and dried over anhydrous Na₂SO₄. After filtration andconcentration, the crude was purified by silica gel columnchromatography (hexane:EtOAc=1:1, R_(f)=0.49) to afford 3 (12.7 g, 15.1mmol, 94%). ¹H NMR (400 MHz, CDCl₃) δ 7.66-7.68 (m, 4H), 7.33-7.43 (m,6H), 3.67-3.73 (m, 1H), 3.57-3.62 (m, 4H), 1.82 (t, J=5.0 Hz, 4H),1.10-1.51 (m, 60H), 1.04 (s, 9H), 0.88 (t, J=6.8 Hz, 6H).

Compound 4: To a solution of 3 (12.6 g, 15.0 mmol) in 1,4-dioxane (220mL), CH₂Cl₂ (70 mL), MeOH (55 mL), and H₂O (55 mL) was added NaIO₄ (7.70g, 36.0 mmol). The reaction mixture was stirred for 16 hours at roomtemperature. The mixture was extracted with Et₂O (500 mL) and H₂O (300mL). The organic layer was separated and dried over anhydrous Na₂SO₄.After filtration and concentration, the crude product was purified bysilica gel column chromatography (Hexane:EtOAc=9:1, R_(f)=0.30) toafford 4 (7.98 g, 14.5 mmol, 97%). Molecular weight for C₃₅H₅₄NaO₃Si(M+Na)⁺ Calc. 573.3740, Found 573.3.

Compound 7: To a solution of 5 (see, Tetrahedron, 63, 1140-1145, 2006;1.09 g, 2.18 mmol) in THF (20 mL) and HMPA (4 mL), LiHMDS (1 M THFsolution, 4.36 mL, 4.36 mmol) was added at −20° C. The resulting mixturewas stirred for 20 minutes at the same temperature, then cooled to −78°C. A solution of 4 (500 mg, 0.908 mmol) in THF (4 mL) was added. Themixture was stirred and allowed to warm to room temperature overnight.MS analysis showed the formation of the di-acid (6; C₅₃H₈₅O₅Si (M−H)⁻calc. 829.6166, observed 829.5). To the mixture, NaHCO₃ (1.10 g, 13.1mmol) and dimethyl sulfate (1.24 mL, 13.1 mmol) were added and stirredfor 2 hours at room temperature. The reaction was quenched by addingsaturated NH₄Cl aqueous solution (50 mL) then extracted with Et₂O (2×100mL). The organic layer was separated and dried over anhydrous Na₂SO₄.After filtration and concentration, the crude product was purified bysilica gel column chromatography (Hexane:EtOAc=9:1, R_(f)=0.35) toafford 7 (270 mg, 0.314 mmol, 35%). Molecular weight for C₅₅H₉₀NaO₅Si(M+Na)⁺ Calc. 881.6455, Found 881.6484.

Compound 8: To a solution of 7 (265 mg, 0.308 mmol) in THF (2.5 mL),n-TBAF (1 M THF solution, 0.555 mL, 0.555 mmol) was added. The reactionmixture was stirred for 14 hours at 45° C. After concentration, themixture was purified by silica gel column chromatography(Hexane:EtOAc=3:1, R_(f)=0.52) to afford 8 (155 mg, 0.250 mmol, 81%).Molecular weight for C₃₉H₇₂NaO₅ (M+Na)⁺ Calc. 643.5277, Found 643.5273.

Compound 9: To a solution of compound 8 (150 mg, 0.242 mmol) and4-(dimethylamino)butyric acid hydrochloride (49 mg, 0.290 mmol) inCH₂Cl₂ (5 mL) were added diisopropylethylamine (0.126 mL, 0.726 mmol),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (56 mg,0.290 mmol) and DMAP (6 mg, 0.0484 mmol). The reaction mixture wasstirred at room temperature for 14 hours. The reaction mixture was thendiluted with CH₂Cl₂ (100 mL) and washed with saturated NaHCO₃ aq. (50mL). The organic layer was dried over MgSO₄, filtered and concentrated.The crude product was purified by silica gel column chromatography (0-5%MeOH in CH₂Cl₂) to afford compound 9 (121 mg, 0.165 mmol, 68%,R_(f)=0.25 developed with 5% MeOH in CH₂Cl₂). Molecular weight forC₄₅H₈₄NO₆ (M+H)⁺ Calc. 734.6299, Found 734.5.

Compound 10: Treatment of compound 9 with CH₃Cl in CH₃CN and CHCl₃ canafford compound 10.

Example 2

Compound 12: To a solution of 11 (Journal of Medicinal Chemistry (1995),38, 636-46; 1.25 g, 2.58 mmol) in THF (20 mL) and HMPA (4 mL), LiHMDS (1M THF solution, 2.58 mL, 2.58 mmol) was added at −20° C. The mixture wasstirred for 20 min at the same temperature, then cooled to −78° C. Asolution of 4 (500 mg, 0.908 mmol) in THF (9 mL) and HMPA (0.9 mL) wasadded. The mixture was stirred from −78° C. to room temperatureovernight. The reaction was quenched by adding H₂O (40 mL) thenextracted with Et₂O (150 mL×3). The organic layer was separated anddried over anhydrous Na₂SO₄. After filtration and concentration, thecrude was purified by silica gel column chromatography(Hexane:EtOAc=9:1, R_(f)=0.35) to give 12 (136 mg, 0.169 mmol, 19%).Molecular weight for C₅₁H₈₂NaO₅Si (M+Na)⁺ Calc. 825.5829, Found 825.5.

Using 13 in place of 5, a procedure analogous to that described forcompound 7 was followed to afford compound 12 (135 mg, 0.168 mmol, 46%).

Compound 15/Compound 16: To a solution of 12 (800 mg, 0.996 mmol) in THF(5 mL), n-TBAF (1 M THF solution, 5 mL, 5.00 mmol) was added. Thereaction mixture was stirred for 16 h at 45° C. After concentration, themixture was purified by silica gel column chromatography to give 15(Hexane:EtOAc=3:1, R_(f)=0.46, 372 mg, 0.659 mmol, 66%) and 16(CH₂Cl₂:MeOH=95:5, R_(f)=0.36, 135 mg, 0.251 mmol, 25%). Molecularweight for 15; C₃₅H₆₄NaO₅ (M+Na)⁺ Calc. 587.4651, Found 587.4652.Molecular weight for 16; C₃₃H₆₁O₅ (M+H)⁺ Calc. 537.4519, Found 537.5.

Compound 17: To a solution of compound 15 (164 mg, 0.290 mmol) and4-(dimethylamino)butyric acid hydrochloride (58 mg, 0.348 mmol) inCH₂Cl₂ (5 mL) were added diisopropylethylamine (0.152 mL, 0.870 mmol),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (67 mg,0.348 mmol) and DMAP (7 mg, 0.058 mmol). The reaction mixture wasstirred at room temperature for 14 hours. The reaction mixture wasdiluted with CH₂Cl₂ (100 mL) and washed with saturated NaHCO₃ aq. (50mL). The organic layer was dried over MgSO₄, filtered and concentrated.The crude was purified by silica gel column chromatography (0-5% MeOH inCH₂Cl₂) to give compound 17 (158 mg, 0.233 mmol, 80%, R_(f)=0.24developed with 5% MeOH in CH₂Cl₂). Molecular weight for C₄₅H₈₄NO₆ (M+H)⁺Calc. 734.6299, Found 734.5.

Compound 18: Treatment of compound 17 with CH₃Cl in CH₃CN and CHCl₃ canafford compound 18.

Compound 19: To a solution of 16 (130 mg, 0.242 mmol) in THF (2 mL) andMeOH (2 mL), trimethylsilyldiazomethane (2 M solution in Et₂O, 0.158 mL,0.315 mmol) was added. The reaction mixture was stirred for 14 h. Afterevaporation, the residue was purified by silica gel columnchromatography (Hexane:EtOAc=3:1, R_(f)=0.50) to give 19 (99 mg, 0.180mmol, 74%). ¹H NMR (400 MHz, CDCl₃) δ 5.29-5.40 (m, 4H), 4.12 (q, J=7.1Hz, 2H), 3.66 (s, 3H), 3.55-3.59 (m, 1H), 2.30 (dd, J=14.7, 7.2 Hz, 4H),1.98-2.07 (m, 8H), 1.60-1.68 (m, 4H), 1.23-1.43 (m, 37H).

Compound 20: To a solution of compound 19 (95 mg, 0.168 mmol) and4-(dimethylamino)butyric acid hydrochloride (42 mg, 0.252 mmol) inCH₂Cl₂ (3 mL) were added diisopropylethylamine (0.088 mL, 0.504 mmol),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (48 mg,0.504 mmol) and DMAP (4 mg, 0.034 mmol). The reaction mixture wasstirred at room temperature for 14 hours. The reaction mixture wasdiluted with CH₂Cl₂ (100 mL) and washed with saturated NaHCO₃ aq. (50mL). The organic layer was dried over MgSO₄, filtered and concentrated.The crude was purified by silica gel column chromatography (0-5% MeOH inCH₂Cl₂) to give compound 20 (103 mg, 0.155 mmol, 92%, R_(f)=0.19developed with 5% MeOH in CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃) δ 5.29-5.40(m, 4H), 4.83-4.89 (m, 1H), 4.12 (q, J=7.1 Hz, 2H), 3.67 (s, 3H),2.28-2.34 (m, 8H), 2.23 (s, 6H), 1.98-2.07 (m, 8H), 1.76-1.83 (m, 2H),1.60-1.68 (m, 4H), 1.23-1.51 (m, 35H).

Compound 21: Treatment of compound 20 with CH₃Cl in CH₃CN and CHCl₃ canafford compound 21.

Example 3 Alternate Synthesis for Di-Aldehyde Intermediate 4

The di-aldehyde 4 can be synthesized as shown in Scheme 3, using1-bromo-9-decene. Di-aldehyde containing a head group 27 can be usefulfor the synthesis of terminal ester-substituted lipids using, e.g., aWittig reaction. Ozonolysis can afford di-aldehyde 4 and 27.

Example 4 Alternate Synthesis for Compound 8

Compound 8 can be synthesized as shown in Scheme 4.

Compound 29: To a stirred suspension of NaH (60% in oil, 82 g, 1.7096mol) in 500 mL anhydrous DMF, a solution of compound 28 (250 g, 1.7096mol) in 1.5 L DMF was added slowly using a dropping funnel at 0° C. Thereaction mixture was stirred for 30 minutes, then benzyl bromide (208.86mL, 1.7096 mol) was added slowly under an atmosphere of nitrogen. Thereaction was then warmed to ambient temperature and stirred for 10hours. The mixture was then quenched with crushed ice (˜2 kg) andextracted with ethyl acetate (2×1 L). The organic layer was washed withwater (1 L) to remove unwanted DMF, dried over Na₂SO₄ and evaporated todryness in vacuo. The crude compound was purified on 60-120 silica gel,eluted with 0-5% MeOH in DCM to afford compound 29 (220 g, 54%) as apale yellow liquid. ¹H NMR (400 MHz, CDCl₃): δ=7.33-7.24 (m, 5H), 4.49(s, 2H), 3.63-3.60 (m, 2H), 3.47-3.43 (m, 2H), 1.63-1.51 (m, 4H),1.39-1.23 (m, 8H).

Compound 30: Compound 29 (133 g, 0.5635 mol) was dissolved in 1.5 L ofDCM, CBr₄ (280.35 g, 0.8456 mol) was added into this stirring solutionand the reaction mixture was cooled to 0° C. under an inert atmosphere.PPh₃ (251.03 g, 0.9571 mol) was then added in portions keeping thetemperature below 20° C. After complete addition, the reaction mixturewas stirred for 3 hours at room temperature. After completion of thereaction, the solid (PPh₃O) that precipitated from the reaction mixturewas removed by filtration, and the filtrate was diluted with crushed ice(˜1.5 kg) and extracted with DCM (3×750 mL). The organic layer wasseparated, dried over anhydrous Na₂SO₄ and distilled under vacuum. Theresulting crude compound was chromatographed on 60-120 mesh silica gelcolumn using 0-5% ethyl acetate in hexanes as eluting system to affordcompound 30 (150 g, 89%) as pale yellow liquid. ¹H NMR (400 MHz, CDCl₃):δ=7.33-7.25 (m, 5H), 4.49 (s, 2H), 3.47-3.41 (m, 2H), 3.41-3.37 (m, 2H),1.86-1.80 (m, 4H), 1.62-1.56 (m, 2H), 1.42-1.29 (m, 8H).

Compound 31: To freshly activated Mg turnings (24.08 g, 1.003 mol) wasadded 200 mL anhydrous THF, followed by the addition of pinch of iodineinto the mixture under an inert atmosphere. A solution of Compound 30(150 g, 0.5016 mol) in 1 L of dry THF was added slowly, controlling theexothermic reaction. The reaction was then heated to reflux for 1 hour,then cooled to room temperature. Methyl formate (60.24 g, 1.0033 mol)was then added slowly and the reaction was continued for 2 hours. Aftercompletion, the reaction was quenched by slow addition of 10% HClfollowed by water (1 L) and extracted with ethyl acetate (3×1 L). Theorganic layer was taken in 5 litre beaker, diluted with 500 mL ofmethanol and cooled to 0° C. To this solution, an excess of NaBH₄ (˜5eq) was added in portions to ensure hydrolysis of the formate esterwhich was not cleaved by addition of HCl. The resulting solution wasstirred for an hour and then volatilites were removed under vacuum. Theresidue was taken in water (1 L) and acidified by 10% HCl solution (pH4). The product was then extracted with ethyl acetate (3×1 L). theorganic phase was then dried and concentrated on rotary evaporator toafford the desired compound 31 (57 g, 24%) as solid. ¹H NMR (400 MHz,CDCl₃): δ=7.35-7.32 (m, 8H), 7.29-7.24 (m, 2H), 4.49 (s, 4H), 3.56 (m,1H), 3.46-3.43 (m, 4H), 1.63-1.56 (m, 4H), 1.44-1.34 (m, 28H). ¹³C NMR(100 MHz, CDCl₃): δ=138.56, 128.21, 127.49, 127.34, 72.72, 71.76, 70.37,37.37, 29.64, 29.56, 29.47, 29.33, 26.07, 25.54.

Compound 32: Compound 31 (56 g, 0.1196 mol) was dissolved in 700 mL dryTHF and cooled to 0° C. TBSCl (36.06 g, 0.2396 mol) was added slowlyfollowed by the addition of imidazole (32.55 g, 0.4786 mol) under aninert atmosphere. The reaction was then stirred at room temperature for18 hours. Upon completion, the reaction was quenched with ice (˜1 kg)and extracted with ethyl acetate (3×500 mL). The organic layer wasseparated, washed with saturated NaHCO₃ solution to remove acidicimpurities, dried over Na₂SO₄ and evaporated under reduce pressure toafford a crude compound that was purified by silica gel (60-120 mesh)and eluted with 0-10% ethyl acetate hexane to afford (60 g, 82%) ofcompound 32 as yellowish oil. ¹H NMR (400 MHz, CDCl₃): δ=7.33-7.24 (m,10H), 4.49 (s, 4H), 3.60-3.57 (m, 1H), 3.46-3.43 (m, 4H), 1.61-1.54 (m,4H), 1.41-1.26 (m, 28H), 0.87 (s, 9H), 0.02 (s, 6H).

Compound 33: Compound 32 (60 g, 0.1030 mol) was dissolved in 500 mLethyl acetate and degassed with N₂ for 20 minutes. (10 wt %) Pd oncarbon (12 g) was added and the reaction was stirred under an atmosphereof hydrogen for 18 hours. After completion, the mixture was filteredthrough a bed of celite and washed with ethyl acetate. The filtrate wasevaporated under vacuum to afford compound 33 (19 g, 46%) that was pureenough to use in the next synthetic sequence. ¹H NMR (400 MHz, CDCl₃):δ=3.64-3.58 (m, 5H), 1.59 (br, 2H), 1.57-1.51 (m, 4H), 1.38-1.22 (m,28H), 0.87 (s, 9H), 0.02 (s, 6H).

Compound 34: Compound 33 (8.2 g, 0.0199 mol) was dissolved in 100 mL dryDCM and cooled to 0° C. TEA (22.14 mL, 0.1592 mol) was added under aninert atmosphere. After stirring the mixture for 5 minutes, mesylchloride (4.6 mL, 0.059 mol) was added drop wise and the reaction wasstirred further for 3 hours. After completion of the reaction, themixture was quenched with ice (˜200 g) and extracted with DCM (3×75 mL).The organic layer was dried over anhydrous sodium sulfate and evaporatedto afford a crude compound which was purified on a 60-120 mesh silicagel column using 0-30% ethyl acetate in hexane as eluting system toafford compound 34 (8.2 g, 73%) as a pale yellow liquid. ¹H NMR (400MHz, CDCl₃): δ=4.22-4.19 (m, 4H), 3.60-3.58 (m, 1H), 2.99 (s, 6H),1.75-1.69 (m, 4H), 1.38-1.28 (m, 28H), 0.86 (s, 9H), 0.02 (s, 6H).

Compound 35: To a solution of compound 34 (8.2 g, 0.0146 mol) in 400 mLdry ether was added MgBr₂.Et₂O (22.74 g, 0.08817 mol) in portions at 0°C. under a nitrogen atmosphere. After complete addition, the reactionmixture was heated to reflux for 28 hours. After completion of reaction,inorganic material formed in the reaction was removed by filtration. Thefiltrate was evaporated and the resulting crude compound was purified on60-120 mesh silica gel column using 0-3% ethyl acetate in hexanes aseluting system to afford compound 35 (6.6 g, 85%) as a colorless liquid.¹H NMR (400 MHz, CDCl₃): δ=3.61-3.58 (m, 1H), 3.41-3.37 (t, 4H, J=6.8Hz), 1.87-1.80 (m, 4H), 1.42-1.25 (m, 24H), 0.87 (s, 9H), 0.012 (s, 6H).

Compound 36: A solution of ethynyl trimethyl silane (5.3 mL, 0.0378 mol)in 60 mL dry THF was cooled to −78° C. and 1.4 M n-BuLi (23 mL, 0.03405mol) in hexane was added slowly under an inert atmosphere. The reactionwas stirred for 10 minutes, then HMPA (2.3 g, 0.01324 mol) was added andthe resulting mixture was then stirred for 2 hours at 0° C., then cooledto −78° C. To this a solution of compound 35 (5 g, 0.0094 mol) in 60 mLdry THF was added slowly and after complete addition, the reaction waswarmed to room temperature and maintained for 18 hours. The reactionprogress was monitored by ¹H NMR. After completion, the reaction mixturewas cooled to 0° C. and quenched by careful addition of saturated NH₄Clsolution (50 mL) followed by water (200 mL). The aqueous phase wasextracted with hexane (3×250 mL). The organic layer was dried andsolvent removed under vacuum to afford compound 36 (5 g, 94%), which wasused without further purification. ¹H NMR (400 MHz, CDCl₃): δ=3.62-3.56(m, 1H), 2.21-2.17 (m, 4H), 1.49-1.47 (m, 4H), 1.37-1.26 (m, 24H), 0.87(s, 9H), 0.13 (s, 18H), 0.021 (s, 6H).

Compound 37: To a stirred solution of compound 36 (5 g, 0.0088 mol) in50 mL methanol, was added K₂CO₃ (6.1 g, 0.044 mol) in one portion, andthe resulting mixture was stirred for 18 hours at ambient temperature.Volatilities were then removed on a rotary evaporator and the crudemixture was diluted with 100 mL water and extracted with hexane (3×100mL). The organic layer was dried over Na₂SO₄ and evaporated under vacuumto afford compound 37 (3.5 g, 97%) which was used which was used withoutfurther purification. ¹H NMR (400 MHz, CDCl₃): δ=3.60-3.58 (m, 1H),2.19-2.14 (m, 4H), 1.93-1.92 (m, 2H), 1.54-1.49 (m, 4H), 1.37-1.27 (m,24H), 0.87 (s, 9H), 0.02 (s, 6H).

Compound 39: Compound 37 (2.5 g, 0.00598 mol) was dissolved in 25 mL dryTHF and cooled to −40° C. n-BuLi (1.4 M in hexane 12.9 mL, 0.01794 mol)was added slowly, followed, after a 10 minute interval, by slow additionof HMPA (25 mL). The resulting mixture was maintained for 30 minutes−40° C. under a nitrogen atmosphere. A solution of compound 38 (3.5 g,1.01196 mol) in 25 mL dry THF was then added drop wise to the cooledreaction mixture. The resulting mixture was warmed to room temperatureover 2 hours, then stirred at room temperature for 18 hours. The mixturewas then quenched by adding saturated NH₄Cl solution (˜50 mL) and theproduct was extracted with ethyl acetate (3×50 mL). The solvent wasremoved on a rotary evaporator and the resulting crude product waspurified by (100-200 mesh) silica gel column using 0-3% ethyl acetate indichloromethane as eluting system to afford compound 39 (0.9 g, 18%) asa yellow oil. ¹H NMR (400 MHz, CDCl₃): δ=4.56-4.55 (m, 2H), 3.87-3.83(m, 2H), 3.74-3.68 (m, 2H), 3.59-3.57 (m, 1H), 3.49-3.46 (m, 2H),3.39-3.33 (m, 2H), 2.13-2.10 (m, 8H), 1.87-1.75 (m, 2H), 1.74-1.66 (m,2H), 1.57-1.42 (m, 20H), 1.40-1.19 (m, 40H), 0.87 (s, 9H), 0.02 (s, 6H).

Compound 40: To a solution of compound 39 (504 mg, 0.598 mmol) in 10 mLdry ether was added MgBr₂.Et₂O (926 mg, 3.59 mmol). The reaction mixturewas stirred for 14 hours, then quenched by adding saturated NaHCO₃aqueous solution. The product was extracted with CH₂Cl₂. The organiclayer was dried over Na₂SO₄, filtered and concentrated. The crudeproduct was purified by silica gel column chromatography to affordcompound 40 (307 mg, 0.455 mmol, 76%, R_(f)=0.36 developed withhexane:EtOAc=2:1). ¹H NMR (400 MHz, CDCl₃) δ 3.59-3.66 (m, 5H), 2.14 (t,J=6.6 Hz, 8H), 1.21-1.59 (m, 52H), 0.88 (s, 9H), 0.03 (s, 6H).

Compound 41: To a stirred solution of 40 (180 mg, 0.267 mmol) inanhydrous DMF (5 mL) was added pyridinium dichromate (603 mg, 1.60mmol). The reaction mixture was stirred for 48 hours. After dilutionwith water (20 mL), the mixture was extracted with Et₂O (3×40 mL). Theorganic layer was dried over Na₂SO₄, filtered and concentrated. Thecrude product was purified by silica gel column chromatography to affordcompound 41 (53 mg, 0.075 mmol, 28%, R_(f)=0.25 developed withCH₂Cl₂:MeOH:AcOH=95:4.5:0.5). Molecular weight for C₄₃H₇₇O₅S (M−H)⁻Calc. 701.5540, Found 701.5. This compound can be synthesized by TEMPOoxidation.

Compound 42: A procedure analogous to that described for compound 19afforded compound 42 (23 mg 0.032 mmol, 21% from compound 40). ¹H NMR(400 MHz, CDCl₃) δ 3.67 (s, 6H), 3.59-3.62 (m, 1H), 2.30 (t, J=7.5 Hz,4H), 2.13 (t, J=6.8 Hz, 8H), 1.27-1.64 (m, 48H), 0.88 (s, 9H), 0.03 (s,6H).

Reduction using P-2 nickel conditions can give compound 43 andsubsequent deprotection by TBAF can afford compound 8.

Example 5 Alternate Synthesis for Compound 8

Compound 8 can be synthesized as shown in Scheme 5. The bromide 51 canbe converted to its Grignard reagent then coupled with ethyl formate toafford compound 52. Subsequent acid treatment, oxidation, and reductioncan give compound 8.

Example 6 Alternate Synthesis for Compound 8

Compound 8 can be synthesized as shown in Scheme 6. Either bromides ofcompound 58, 60, or 62 can be reacted with ethyl formate to generateterminal-functionalized di-olefin chain. Compound 8 can then be preparedfrom the diolefin chain compounds using standard chemical reactions.

Example 7 General Synthetic Scheme for Terminal Ester Lipids

As shown in Scheme 7, chain length and linker length as well as alkylgroups in ester functionality and substituents on nitrogen atom can bederivatized.

Example 8 General Synthetic Scheme 2 for Terminal Ester Lipids

As shown in Scheme 8, copper-mediated coupling affords di-yne containinglipid chain with terminal functional groups, which can be easily reducedto generate di-ene containing lipid chains. The length of linker andlipid chain as well as functional substituent groups (R, R₁, R₂) can bederivatized.

Example 9 Synthesis of Terminal Benzyl Ester Lipid

Compound 201: Compound 7 (1.30 g, 1.51 mmol) was treated with lithiumhydroxide monohydrate (317 mg, 7.55 mmol) in THF (25 mL) and H₂O (5 mL)for 12 h. Amberlite IR-120 (plus) ion exchange resin was added thenstirred for 10 minutes. The resulting clear solution was filtered,washed with THF/H₂O and evaporated. Co-evaporation with toluene gave thecompound 201 (1.22 g, 1.47 mmol, 97%). Molecular weight for C₅₃H₈₅O₅Si(M−H)⁻ Calc. 829.6166, Found 829.5.

Compound 202: A procedure analogous to that described for compound 9 wasfollowed with benzylalcohol and 201 (101 mg, 0.121 mmol) to affordcompound 202 (87 mg, 0.0860 mmol, 71%). ¹H NMR (400 MHz, CDCl₃) δ7.68-7.66 (m, 4H), 7.42-7.30 (m, 16H), 5.38-5.30 (m, 4H), 5.11 (s, 4H),3.71-3.68 (m, 1H), 2.35 (t, J=7.6 Hz, 4H), 2.04-1.97 (m, 8H), 1.66-1.62(m, 4H), 1.40-1.07 (m, 44H), 1.04 (s, 9H).

Compound 203: A procedure analogous to that described for compound 8 wasfollowed with 202 (342 mg, 0.338 mmol) to afford compound 202 (136 mg,0.176 mmol, 52%). ¹H NMR (400 MHz, CDCl₃) δ 7.38-7.30 (m, 10H),5.38-5.30 (m, 4H), 5.11 (s, 4H), 3.57 (brs, 1H), 2.35 (t, J=7.6 Hz, 4H),2.01-1.98 (m, 8H), 1.66-1.60 (m, 4H), 1.45-1.25 (m, 44H).

Compound 204: A procedure analogous to that described for compound 9 wasfollowed with 203 (133 mg, 0.172 mmol) to afford compound 204 (93 mg,0.105 mmol, 61%). ¹H NMR (400 MHz, CDCl₃) δ 7.38-7.26 (m, 10H),5.38-5.30 (m, 4H), 5.11 (s, 4H), 4.88-4.83 (m, 1H), 2.37-2.27 (m, 8H),2.22 (s, 6H), 2.03-1.97 (m, 8H), 1.81-1.26 (m, 50H).

Example 10 Synthesis of Terminal t-Butyl Ester Lipid and the Derivatives

Compound 206: A procedure analogous to that described for compound 12was followed with 205 (3.80 g, 0.7.61 mmol) and 4 (1.75 g, 3.17 mmol) toafford compound 206 (2.00 g, 2.12 mmol, 67%). ¹H NMR (400 MHz, CDCl₃) δ7.68-7.66 (m, 4H), 7.42-7.33 (m, 6H), 5.39-5.31 (m, 4H), 3.71-3.68 (m,1H), 2.20 (t, J=7.6 Hz, 4H), 2.01-1.98 (m, 8H), 1.59-1.55 (m, 4H), 1.44(s, 18H), 1.41-1.11 (m, 44H), 1.04 (s, 9H).

Compound 207: A procedure analogous to that described for compound 8 wasfollowed with 206 (265 mg, 0.281 mmol) to afford compound 207 (161 mg,0.228 mmol, 81%). ¹H NMR (400 MHz, CDCl₃) δ 5.38-5.30 (m, 4H), 3.58(brs, 1H), 2.20 (t, J=7.4 Hz, 4H), 2.01-1.98 (m, 8H), 1.59-1.55 (m, 4H),1.44 (s, 18H), 1.35-1.26 (m, 44H).

Compound 208: A procedure analogous to that described for compound 9 wasfollowed with 207 (158 mg, 0.224 mmol) to afford compound 208 (138 mg,0.169 mmol, 75%). Molecular weight for C₅₁H₉₆NO₆ (M+H)⁺ Calc. 818.7238,Found 818.7.

Compound 209: Compound 208 (148 mg, 0.181 mmol) was treated with TFA(1.5 mL) in CH₂Cl₂ (6 mL) for 2.5 h. After evaporation andco-evaporation with toluene gave the compound 209 (154 mg, quant.).Molecular weight for C₄₃H₈₀NO₆ (M+H)⁺ Calc. 706.5980, Found 706.5.

Compound 210: A procedure analogous to that described for compound 9 wasfollowed with 209 (0.061 mmol) and cis-2-Hexen-1-ol (18.3 mg, 0.183mmol) to afford compound 210 (32 mg, 0.0368 mmol, 60%). Molecular weightfor C₅₅H₁₀₀NO₆ (M+H)⁺ Calc. 870.7551, Found 870.5.

Example 11 Synthesis of Internal Ester/Amide Lipids-1

Compound 213: Compound 211 (503 mg, 1.0 mmol) was treated with 212 (533mg, 3.0 mmol) in CH₂Cl₂ (35 mL) and DIPEA (1.74 mL, 10 mmol) for 14 h.Aqueous work-up then column chromatography gave compound 213 (506 mg,0.748 mmol, 75%). Molecular weight for C₄₁H₇₈N₃O₄ (M+H)⁺ Calc. 676.5992,Found 676.4.

Compound 215: Compound 211 (503 mg, 1.0 mmol) was treated with 214 (469mg, 3.0 mmol) and K₂CO₃ (1.38 g, 10 mmol) in CH₂Cl₂ (35 mL) for 14 h.Aqueous work-up then column chromatography gave compound 215 (244 mg,0.346 mmol, 35%). Molecular weight for C₄₃H₈₀NO₆ (M+H)⁺ Calc. 706.5986,Found 706.4.

Compound 217: Compound 211 (425 mg, 0.845 mmol) was treated with 216(525 mg, 3.08 mmol) and K₂CO₃ (1.17 g, 8.45 mmol) in CH₂Cl₂ (35 mL) for14 h. Aqueous work-up then column chromatography gave compound 217 (407mg, 0.554 mmol, 66%). Molecular weight for C₄₅H₈₄NO₆ (M+H)⁺ Calc.734.6299, Found 734.4.

Compound 219: Compound 211 (503 mg, 1.0 mmol) was treated with 218 (595mg, 3.0 mmol) and K₂CO₃ (1.38 g, 10 mmol) in CH₂Cl₂ (35 mL) for 14 h.Aqueous work-up then column chromatography gave compound 219 (519 mg,0.657 mmol, 66%). Molecular weight for C₄₉H₉₂NO₆ (M+H)⁺ Calc. 790.6925,Found 790.7.

Example 12 Synthesis of Internal Ester Lipid-223

Compound 221: A procedure analogous to that described for compound 9 wasfollowed with 220 (390 mg, 1.93 mmol) and 218 (765 mg, 3.86 mmol) toafford compound 221 (878 mg, 1.56 mmol, 81%). ¹H NMR (400 MHz, CDCl₃) δ5.67-5.61 (m, 2H), 5.54-5.48 (m, 2H), 4.62 (d, J=6.8 Hz, 4H), 2.47 (t,J=7.2 Hz, 4H), 2.33 (t, J=7.2 Hz, 4H), 2.12-2.06 (m, 4H), 1.93-1.86 (m,4H), 1.38-1.26 (m, 32H), 0.88 (t, J=6.8 Hz, 6H).

Compound 222: Compound 221 (318 mg, 0.565 mmol) was treated withNaBH(OAc)₃ (360 mg, 1.70 mmol) in CH₂Cl₂ (5 mL) and AcOH (0.2 mL) for 16h. After evaporation, column chromatography gave compound 222 (141 mg,0.250 mmol, 44%). Molecular weight for C₃₅H₆₅O₅ (M+H)⁺ Calc. 565.4832,Found 565.4.

Compound 223: A procedure analogous to that described for compound 9 wasfollowed with 222 (137 mg, 0.243 mmol) to afford compound 223 (137 mg,0.202 mmol, 83%). Molecular weight for C₄₁H₇₆NO₆ (M+H)⁺ Calc. 678.5673,Found 678.5.

Example 13 Synthesis of Internal Ester Lipid-227

Compound 225: A procedure analogous to that described for compound 9 wasfollowed with 224 (200 mg, 0.774 mmol) and 216 (264 mg, 1.55 mmol) toafford compound 225 (341 mg, 0.606 mmol, 78%). Molecular weight forC₃₅H₆₂NaO₅ (M+Na)⁺ Calc. 585.4495, Found 585.5.

Compound 226: Compound 225 (283 mg, 0.503 mmol) was treated with NaBH₄(57 mg, 1.51 mmol) in THF (5 mL) and AcOH (0.2 mL) for 8 h. Afterevaporation, column chromatography gave compound 226 (185 mg, 0.328mmol, 65%). Molecular weight for C₃₅H₆₄NaO₅ (M+Na)⁺ Calc. 587.4651,Found 587.3.

Compound 227: A procedure analogous to that described for compound 9 wasfollowed with 226 (230 mg, 0.407 mmol) to afford compound 227 (248 mg,0.366 mmol, 90%). Molecular weight for C₄₁H₇₆NO₆ (M+H)⁺ Calc. 678.5673,Found 678.5.

Example 14 Synthesis of Terminal Ester Lipid with Linoleyl Chain-232

Compound 230: A procedure analogous to that described for compound 7 wasfollowed with 228 (3.27 g, 6.0 mmol) and 4 (1.27 g, 2.30 mmol) to affordcompound 230 (1.31 g, 1.53 mmol, 67%). ¹H NMR (400 MHz, CDCl₃) δ7.68-7.66 (m, 4H), 7.42-7.33 (m, 6H), 5.42-5.29 (m, 8H), 3.71-3.68 (m,1H), 3.66 (s, 6H), 2.77 (t, J=5.8 Hz, 4H), 2.33-2.28 (m, 4H), 2.11-2.01(m, 8H), 1.69-1.60 (m, 4H), 1.43-1.10 (m, 32H), 1.04 (s, 9H).

Compound 231: A procedure analogous to that described for compound 8 wasfollowed with 230 (1.30 g, 1.52 mmol) to afford compound 231 (611 mg,0.990 mmol, 65%). ¹H NMR (400 MHz, CDCl₃) δ 5.41-5.29 (m, 8H), 3.67 (s,6H), 3.58 (brs, 1H), 2.77 (t, J=5.8 Hz, 4H), 2.32 (t, J=7.4 Hz, 4H),2.10-2.00 (m, 8H), 1.69-1.60 (m, 4H), 1.43-1.29 (m, 32H).

Compound 232: A procedure analogous to that described for compound 9 wasfollowed with 231 (520 mg, 0.843 mmol) to afford compound 232 (600 mg,0.822 mmol, 97%). Molecular weight for C₄₅H₈₀NO₆ (M+H)⁺ Calc. 730.5986,Found 730.5.

Example 15 Synthesis of Terminal Ester Lipid with Linoleyl Chain-232

Compound 231 was also synthesized as shown Scheme 15.

Compound 112: Compound 111 (840 mg, 2.69 mmol) was treated with dimethyl(1-diazo-2-oxopropyl)phosphonate (0.970 mL, 6.46 mmol) and K₂CO₃ (1.49g, 10.8 mmol) in MeOH (40 mL) for 6 h. Aqueous work-up then columnchromatography gave compound 112 (700 mg, 2.30 mmol, 86%). ¹H NMR (400MHz, CDCl₃) δ 3.58 (brs, 1H), 2.18 (td, J=7.1, 2.6 Hz, 4H), 1.94 (t,J=2.6 Hz, 2H), 1.56-1.25 (m, 28H).

Compound 234: Compound 112 (207 mg, 0.680 mmol) was treated with 233(316 mg, 1.36 mmol), K₂CO₃ (282 mg, 2.04 mmol), NaI (408 mg, 2.72 mmol)and CuI (518 mg, 2.72 mmol) in DMF (3.5 mL) for 18 h. Aqueous work-upthen column chromatography gave compound 234 (292 mg, 0.480 mmol, 71%).Molecular weight for C₃₉H₆₁O₅ (M+H)⁺ Calc. 609.4519, Found 609.5.

Compound 231: To a stirred solution of nickel(II) acetate tetrahydrate(533 mg, 2.14 mmol) in EtOH (28.5 mL), 1 M solution of NaBH₄ in EtOH(2.14 mL) was added at room temperature. After 30 min, ethylenediamine(0.574 mL, 8.57 mmol) and a solution of 234 (290 mg, 0.476 mmol) in EtOH(3 mL) was added then stirred for 1 h. The reaction mixture was filteredthrough Celite and evaporated. Aqueous work-up then columnchromatography gave compound 231 (219 mg, 0.355 mmol, 75%). Molecularweight for C₃₉H₆₉O₅ (M+H)⁺ Calc. 617.5145, Found 617.3.

Example 16 Synthesis of Internal Oxime Lipid-238

Compound 237: Compound 235 (465 mg, 1.78 mmol) was treated withhydrazine monohydrate (64-65%, 0.135 mL, 1.78 mmol) in EtOH (15 mL) for4 h. After filtration then evaporation, the crude was re-suspended inEtOH (5 mL). To this solution was added compound 111 (160 mg, 0.512mmol) and AcOH (a few drops). Aqueous work-up then column chromatographygave compound 237 (165 mg, 0.306 mmol, 60%). Molecular weight forC₃₃H₆₇N₂O₃ (M+H)⁺ Calc. 539.5152, Found 539.3.

Compound 238: A procedure analogous to that described for compound 9 wasfollowed with 237 (162 mg, 0.301 mmol) to afford compound 238 (168 mg,0.258 mmol, 86%). Molecular weight for C₃₉H₇₈N₃O₄ (M+H)⁺ Calc. 652.5992,Found 652.4.

Example 17

8-benzyloxy-octan-1-ol (240)

To a stirred suspension of NaH (60% in oil, 82 g, 1.7096 mol) in 500 mLanhydrous DMF, a solution of compound 239 (250 g, 1.7096 mol) in 1.5 LDMF was added slowly using a dropping funnel at 0° C. The reactionmixture was stirred for 30 minutes, then benzyl bromide (208.86 mL,1.7096 mol) was added slowly under a nitrogen atmosphere. The reactionwas then warmed to ambient temperature and stirred for 10 hours. Aftercompletion of reaction, the mixture was quenched with crushed ice (˜2kg) and extracted with ethyl acetate (2×1 L). The organic layer washedwith water (1 L) to remove unwanted DMF, dried over Na₂SO₄ andevaporated to dryness under vacuum. The crude compound was purified on60-120 silica gel, eluted with 0-5% MeOH in DCM to afford compound 240(220 g, 54%) as pale yellow liquid. H¹ NMR (400 MHz, CDCl₃): δ=7.33-7.24(m, 5H), 4.49 (s, 2H), 3.63-3.60 (m, 2H), 3.47-3.43 (m, 2H), 1.63-1.51(m, 4H), 1.39-1.23 (m, 8H).

(8-bromo-octyloxymethyl)-benzene (241)

Compound 240 (133 g, 0.5635 mol) was dissolved in 1.5 L of DCM, CBr₄(280.35 g, 0.8456 mol) was added to this stirring solution and thereaction mixture was cooled to 0° C. under an inert atmosphere. PPh₃(251.03 g, 0.9571 mol) was then added in portions maintaining thetemperature below 20° C. and after complete addition, the reactionmixture was stirred for 3 hours at room temperature. After completion ofreaction, solid (PPh₃O) precipitated out from the reaction mixture wasisolated by filtration and the filtrate was diluted with crushed ice(˜1.5 kg) and extracted with DCM (3×750 mL). The organic layer wasseparated, dried over anhydrous Na₂SO₄ and distilled under vacuum. Theresulting crude compound was chromatographed on 60-120 mesh silica gelcolumn using 0-5% ethyl acetate in hexanes as eluting system to affordcompound 241 (150 g, 89%) as pale yellow liquid. ¹H NMR (400 MHz,CDCl₃): δ=7.33-7.25 (m, 5H), 4.49 (s, 2H), 3.47-3.41 (m, 2H), 3.41-3.37(m, 2H), 1.86-1.80 (m, 4H), 1.62-1.56 (m, 2H), 1.42-1.29 (m, 8H).

1,17-bis-benzyloxy-heptadecan-9-ol (242)

To freshly activated Mg turnings (24.08 g, 1.003 mol) was added 200 mLanhydrous THF, followed by the addition of pinch of iodine into themixture under inert atmosphere. After initiation of the Grignardformation a solution of Compound 241 (150 g, 0.5016 mol) in 1 L of dryTHF was added slowly controlling the exothermic reaction. After completeaddition, the reaction was heated to reflux for 1 hour, then cooled toroom temperature. Methyl formate (60.24 g, 1.0033 mol) was then addedslowly and reaction was continued for 2 hours. After completion, thereaction was quenched by slow addition of 10% HCl followed by water (1L) and extracted with ethyl acetate (3×1 L). The organic layer was takenin 5 litre beaker, diluted with 500 mL of methanol and cooled to 0° C.To this solution excess of NaBH₄ (˜5 eq) was added in portions to ensurethe hydrolysis of formate ester which was not cleaved by addition ofHCl. The resulting solution was stirred for an hour and then volatiliteswere removed under vacuum. The residue was taken in water (1 L) andacidified by 10% HCl solution (P^(H) 4). The product was then extractedwith ethyl acetate (3×1 L). The organic phase was then dried andconcentrated on rotary evaporator to afford compound 242 (57 g, 24%) assolid. ¹H NMR (400 MHz, CDCl₃): δ=7.35-7.32 (m, 8H), 7.29-7.24 (m, 2H),4.49 (s, 4H), 3.56 (m, 1H), 3.46-3.43 (m, 4H), 1.63-1.56 (m, 4H),1.44-1.34 (m, 28H). C¹³ NMR (100 MHz, CDCl₃): δ=138.56, 128.21, 127.49,127.34, 72.72, 71.76, 70.37, 37.37, 29.64, 29.56, 29.47, 29.33, 26.07,25.54.

[9-benzyloxy-1-(8-benzyloxy-octyl)-nonyloxy]-cert-butyl-dimethyl-silane(243)

Compound 242 (56 g, 0.1196 mol) was dissolved in 700 mL of anhydrous THFand cooled to 0° C. TBMS-Cl (36.06 g, 0.2396 mol) was added slowlyfollowed by addition of imidazole (32.55 g, 0.4786 mol) under an inertatmosphere. The reaction was then stirred at room temperature for 18hours, then quenched with ice (˜1 kg). The product was extracted withethyl acetate (3×500 mL). The organic layer was separated, washed withsaturated NaHCO₃ solution to remove the acidic impurity, dried overNa₂SO₄ and evaporated under reduce pressure to obtain crude compoundwhich was purified by silica gel (60-120 mesh) and eluted with 0-10%ethyl acetate hexane to afford (60 g, 82%) of compound 243 as yellowishoil. H¹ NMR (400 MHz, CDCl₃): δ=7.33-7.24 (m, 10H), 4.49 (s, 4H),3.60-3.57 (m, 1H), 3.46-3.43 (m, 4H), 1.61-1.54 (m, 4H), 1.41-1.26 (m,28H), 0.87 (s, 9H), 0.02 (s, 6H)

9-(tert-butyl-dimethyl-silanyloxy)-heptadecane-1,17-diol (244)

Compound 243 (60 g, 0.1030 mol) was dissolved in 500 mL ethyl acetateand degassed with N₂ for 20 min. (10 wt %) Pd on carbon (12 g) was addedand reaction was stirred under an atmosphere of hydrogen for 18 hours.After completion, the mixture was filtered through a bed of celite andwashed with ethyl acetate. The filtrate was evaporated under vacuum.Compound 244 (19 g, 46%) thus obtained was pure enough to carry out thenext reaction. ¹H NMR (400 MHz, CDCl₃): δ=3.64-3.58 (m, 5H), 1.59 (br,2H), 1.57-1.51 (m, 4H), 1.38-1.22 (m, 28H), 0.87 (s, 9H), 0.02 (s, 6H).

9-(tert-butyl-dimethyl-silanyloxy)-heptadecanedioic acid (245)

To a stirred solution of 244 (2 g, 0.0049 mol) in anhydrous DMF (40 mL)was added pyridinium dichromate (2.7 g, 0.0074 mol) at 0° C. under aninert atmosphere. The reaction mixture was then allowed to warm to roomtemperature over a period of 10-15 minutes and continued for 24 hours.Then, the reaction was diluted with water (100 mL). The aqueous phasewas extracted using DCM (3×40 mL). The organic phase was washed withbrine (1×25 mL) and concentrated under vacuum to afford crude acid whichwas then purified by (100-200 mesh) silica gel column using 0-30% ethylacetate in hexanes system. Pure product (245) was obtained (0.7 g, 33%)as a pale yellow oil. ¹H NMR (400 MHz, CDCl₃): δ=3.61-3.56 (m, 1H),2.35-2.32 (m, 4H), 1.64-1.59 (m, 4H), 1.40-1.19 (m, 24H), 0.86 (s, 9H),0.017 (s, 6H); LC-MS [M+H] −431.00; HPLC (ELSD) purity—96.94%

Di((Z)-non-2-en-1-yl) 9-((tert-butyldimethylsilyl)oxy)heptadecanedioate(246)

The diacid 245 (0.42 g, 0.97 mmol) was dissolved in 20 mL ofdichloromethane and to it cis-2-nonen-1-ol (0.35 g, 2.44 mmol) was addedfollowed by Hunig's base (0.68 g, 4.9 mmol) and DMAP (12 mg). To thismixture EDCI (0.47 g, 2.44 mmol) was added and the reaction mixture wasstirred at room temperature overnight. The reaction mixture was thendiluted with CH₂Cl₂ (40 mL) and washed with saturated NaHCO₃ (50 mL),water (60 mL) and brine (60 mL). The combined organic layers were driedover anhydrous Na₂SO₄ and solvents were removed in vacuo. The crudeproduct thus obtained was purified by Combiflash Rf purification system(40 g silicagel, 0-10% MeOH in CH₂Cl₂) to afford the pure product 246(0.35 g, 53%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃): δ ¹H NMR (400MHz, CDCl₃) δ 5.64 (dt, J=10.9, 7.4 Hz, 2H), 5.58-5.43 (m, 2H), 4.61 (d,J=6.8 Hz, 4H), 3.71-3.48 (m, 1H), 2.30 (t, J=7.6 Hz, 4H), 2.20-1.98 (m,4H), 1.71-1.53 (m, 4H), 1.31 (ddd, J=8.3, 7.0, 3.7 Hz, 34H), 1.07-0.68(m, 14H), 0.02 (s, 5H). ¹³C NMR (101 MHz, CDCl₃) δ 178.18, 139.81,127.78, 81.73, 81.42, 81.10, 76.72, 64.59, 41.52, 41.32, 38.76, 36.09,34.10, 33.93, 33.80, 33.70, 33.59, 33.55, 33.26, 31.95, 30.34, 29.69,29.58, 29.39, 27.01, 22.56, 18.48, 0.01.

Di((Z)-non-2-en-1-yl) 9-hydroxyheptadecanedioate (247)

The silyl protected diester 246 (0.3 g, 0.44 mmol) was dissolved in 1 Msolution of TBAF in THF (6 mL) and the solution was kept at 40° C. fortwo days. The reaction mixture was diluted with water (60 mL) andextracted with ether (2×50 mL). The combined organic layers wereconcentrated and the thus obtained crude product was purified by columnto isolate the pure product (0.097 g, 39%). ¹H NMR (400 MHz, CDCl₃) δ5.64 (dt, J=10.9, 7.4 Hz, 2H), 5.52 (dt, J=11.0, 6.8 Hz, 2H), 4.61 (d,J=6.8 Hz, 4H), 3.57 (s, 1H), 2.30 (t, J=7.5 Hz, 4H), 2.09 (q, J=7.1 Hz,4H), 1.75-1.53 (m, 4H), 1.53-1.06 (m, 36H), 0.88 (t, J=6.8 Hz, 6H). ¹³CNMR (101 MHz, CDCl₃) δ 173.98, 135.64, 123.57, 77.54, 77.22, 76.91,72.14, 60.41, 37.69, 34.54, 31.89, 29.70, 29.60, 29.44, 29.29, 29.07,27.76, 25.80, 25.15, 22.82, 14.29.

Di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate

The alcohol 247 (0.083 g, 0.147 mmol) was dissolved in 20 mL ofdichloromethane and to it dimethylaminobutyric acid hydrochloride (0.030g, 0.176 mmol) was added followed by Hunig's base (0.045 g, 0.44 mmol)and DMAP (2 mg). To this mixture EDCI (0.034 g, 0.176 mmol) was addedand the reaction mixture was stirred at room temperature overnight andthe TLC (silica gel, 10% MeOH in CH₂Cl₂) showed complete disappearanceof the starting alcohol. The reaction mixture was diluted with CH₂Cl₂(40 mL) and washed with saturated NaHCO₃ (50 mL), water (60 mL) andbrine (60 mL). The combined organic layers were dried over anhyd. Na₂SO₄and solvents were removed in vacuo. The crude product thus obtained waspurified by Combiflash Rf purification system (40 g silicagel, 0-10%MeOH in CH₂Cl₂) to isolate the pure product (0.062 g, 62%) as acolorless oil. ¹H NMR (400 MHz, CDCl₃) δ 5.74-5.58 (m, 2H), 5.51 (dtt,J=9.7, 6.8, 1.3 Hz, 2H), 4.95-4.75 (m, 1H), 4.61 (d, J=6.8 Hz, 4H),2.35-2.24 (m, 8H), 2.22 (d, J=7.9 Hz, 6H), 2.09 (q, J=6.9 Hz, 4H),1.83-1.72 (m, 2H), 1.60 (dd, J=14.4, 7.2 Hz, 4H), 1.49 (d, J=5.7 Hz,4H), 1.41-1.13 (m, 30H), 0.88 (t, J=6.9 Hz, 6H). ¹³C NMR (101 MHz,CDCl₃) δ 173.72, 173.36, 135.40, 123.35, 74.12, 60.18, 58.95, 45.46,34.30, 34.11, 32.45, 31.67, 29.38, 29.35, 29.17, 29.07, 28.84, 27.53,25.28, 24.93, 23.16, 22.59, 14.06. MW calc. for C₄₁H₇₅NO₆ (MH⁺): 678.04,found: 678.5.

Example 18

The following shorter route was used for the synthesis of analogs ofCompound 1 of the present invention The commercial 9-bromonon-1-ene 248was treated with magnesium to form the corresponding Grignard reagentwhich was reacted with ethylformate to give the corresponding adduct 249which on treatment with bromobutyryl chloride to provide the bromoester250. The bromoester 250 on treatment with RuO₄ provided the diacid 251.The bromodiacid 251 on treatment with dimethylamine provided the aminodiacid 252. The diacid 252 on treatment with oxalyl chloride in thepresence of DMF provided the diacid chlorides 253. The lipids 254a-nwere synthesized by treating the acid Chloride 253 with respectivealcohols.

No Starting Alcohol (ROH) 254a

254aS

254aF

254b

254bS

254bF

254bF2

254c

254cS

254cF

254d

254ds

254e

254es

254eF

254f

254fs

254fF

254g

254gs

254h

254hs

254hF

254i

254is

254iF

254j

254js

254jF

254k

254ks

254kS

254l

254lF

254m

254ms

254ns

254os

No Product 254a

254aS

254aF

254b

254bS

254bF

254bF2

254c

254cS

254cF

254d

254ds

254e

254es

254eF

254f

254fs

254fF

254g

254gs

254h

254hs

254hF

254i

254is

254iF

254j

254js

254jF

254k

254ks

254kS

254l

254lF

254m

254ms

254ns

254os

Synthesis of nonadeca-1,18-dien-10-ol (249)

To a flame dried 500 mL RB flask, freshly activated Mg turnings (9 g)were added and the flask was equipped with a magnetic stir bar, anaddition funnel and a reflux condenser. This set-up was degassed andflushed with argon and 100 mL of anhydrous ether was added to the flaskvia syringe. The bromide 3 (51.3 g, 250 mmol) was dissolved in anhydrousether (100 mL) and added to the addition funnel. About 5 mL of thisether solution was added to the Mg turnings while stirring vigorously.An exothermic reaction was noticed (to confirm/accelerate the Grignardreagent formation, 5 mg of iodine was added and immediate decolorizationwas observed confirming the formation of the Grignard reagent) and theether started refluxing. The rest of the solution of the bromide wasadded dropwise while keeping the reaction under gentle reflux by coolingthe flask in water. After the completion of the addition the reactionmixture was kept at 35° C. for 1 hour and then cooled in ice bath. Ethylformate (9 g, 121 mmol) was dissolved in anhydrous ether (100 mL) andtransferred to the addition funnel and added dropwise to the reactionmixture with stirring. An exothermic reaction was observed and thereaction mixture started refluxing. After the initiation of the reactionthe rest of the ethereal solution of formate was quickly added as astream and the reaction mixture was stirred for a further period of 1 hat ambient temperature. The reaction was quenched by adding 10 mL ofacetone dropwise followed by ice cold water (60 mL). The reactionmixture was treated with aq. H₂SO₄ (10% by volume, 300 mL) until thesolution became homogeneous and the layers were separated. The aq. phasewas extracted with ether (2×200 mL). The combined ether layers weredried (Na₂SO₄) and concentrated to afford the crude product which waspurified by column (silica gel, 0-10% ether in hexanes) chromatography.The product fractions were evaporated to provide the pure product 249 asa white solid (30.6 g, 90%). ¹H NMR (400 MHz, CDCl₃) δ 7.26 (s, 1H),5.81 (ddt, J=16.9, 10.2, 6.7 Hz, 8H), 5.04-4.88 (m, 16H), 3.57 (dd,J=7.6, 3.3 Hz, 4H), 2.04 (q, J=6.9 Hz, 16H), 1.59 (s, 1H), 1.45 (d,J=7.5 Hz, 8H), 1.43-1.12 (m, 94H), 0.88 (t, J=6.8 Hz, 2H). ¹³C NMR (101MHz, cdcl₃) δ 139.40, 114.33, 77.54, 77.22, 76.90, 72.21, 37.70, 34.00,29.86, 29.67, 29.29, 29.12, 25.85.

Synthesis of nonadeca-1,18-dien-10-yl 4-bromobutanoate (250)

To a solution of the alcohol 249 (5.6 g, 20 mol) in anhydrous DCM (300mL) was added slowly and carefully Bromobutryl chloride (20 mmol) at 0°C. under inert atmosphere. The reaction mixture was warmed to roomtemperature, stirred for 20 h and monitored by TLC (silica gel, 10%ethyl acetate in hexanes). Upon completion of the reaction, mixture wasdiluted with water (400 mL) and organic layer was separated out. Organicphase was then washed with sat. solution of NaHCO₃ (1×400 mL) followedby brine (1×100 mL) and concentrated under vacuum. Crude product wasthen purified by silica gel (100-200 mesh) column, eluted with 2-3%ethyl acetate in hexane solution to give 6 g (90%) of desired product250 as colorless liquid. ¹H NMR (400 MHz, CDCl₃) δ 5.80 (ddt, J=16.9,10.2, 6.7 Hz, 2H), 5.05-4.81 (m, 5H), 3.46 (t, J=6.5 Hz, 2H), 2.48 (t,J=7.2 Hz, 2H), 2.17 (p, J=6.8 Hz, 2H), 2.11-1.93 (m, 4H), 1.65-1.44 (m,4H), 1.43-1.17 (m, 19H). ¹³C NMR (101 MHz, cdcl₃) δ 172.51, 139.37,114.35, 77.54, 77.23, 76.91, 74.86, 34.31, 33.99, 33.01, 32.96, 29.65,29.56, 29.24, 29.09, 28.11, 25.52.

Synthesis of 9-((4-bromobutanoyl)oxy)heptadecanedioic acid (251)

To a solution of the bromoester 250 (12.1 g, 28.2 mmol) indichloromethane (300 mL) and acetonitrile (300 mL), RuCl₃ (1.16 g, 5 mol%) was added and the mixture was cooled to 10° C. and sodiummetaperiodate (60 g) in water (400 mL) was added dropwise. It wasstirred at 10° C. for 20 hr. The reaction mixture was diluted withwater, The layers were separated and to the organic layer, was addedsaturated brine solution with stirring followed by 3% sodium sulfidesolution drop wise for the decolourisation (dark green to pale yellow).The layers were separated, the organic layer was dried over sodiumsulfate and evaporated at reduced pressure to afford pure product. MWcalcd for C₂₀H₃₅BrO₇ 467.39; Found 465.4 (M−2H). ¹H NMR (400 MHz, DMSO)δ 11.94 (s, 2H), 4.88-4.69 (m, 1H), 3.53 (t, J=6.6 Hz, 2H), 2.43 (t,J=7.2 Hz, 2H), 2.17 (t, J=7.4 Hz, 4H), 2.09-1.95 (m, 2H), 1.90 (s, 3H),1.46 (s, 7H), 1.23 (s, 15H).

Synthesis of 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioic acid(252)

The Bromoacid 251 (2 mmol) is dissolved in 2M solution of dimethylaminein THF (20 mL) and to it 1 g of anhudrous K₂CO₃ was added and themixture was heated in a pressure bottle at 50° C. overnight. The TLCshowed the completion of the reaction. The reaction mixture wasacidified with acetic acid and diluted with water (100 mL) and extractedwith dichloromethane (2×60 mL). The combined organic layers wereconcentrated dried and used as such in the next reaction. MW calcd forC₂₃H₄₃NO₆ 429.59; Found 430.6 (MH)⁺. ¹H NMR (400 MHz, DMSO) δ11.87-11.82 (m, 7H), 5.75 (d, J=0.7 Hz, 15H), 4.85-4.69 (m, 38H),3.64-3.55 (m, 12H), 3.35-2.83 (m, 106H), 3.01-2.90 (m, 59H), 2.94 (ddd,J=30.6, 7.7, 4.0 Hz, 63H), 2.90-2.73 (m, 9H), 2.70 (s, 221H), 2.57-2.46(m, 91H), 2.44-2.30 (m, 76H), 2.17 (t, J=7.3 Hz, 147H), 1.89 (tq,J=15.5, 7.6 Hz, 88H), 1.79-1.69 (m, 13H), 1.65-1.32 (m, 311H), 1.28 (d,J=46.0 Hz, 598H).

Synthesis of 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioyl chloride(253)

The diacid 252 is converted to the corresponding diacid chloride 253 bytreating it with oxalyl chloride in dichloromethane in the presence ofcatalytic DMF and the crude acid chloride obtained after theconcentration of the reaction mixture was used as such for the couplingwith different alcohols.

General Procedure for the Synthesis of Cationic Lipids 254a-n

To a solution of the acid chloride 253 (500 mg, 1 mmol) indichloromethane (30 mL) the corresponding alcohol (5 equivalent) wasadded at room temperature followed by solid K₂CO₃ (1 g) and the solutionwas stirred for 16 h at room temperature. The reaction mixture wasdiluted with dichloromethane (100 mL) and washed with satd. NaHCO₃ (100mL) and the organic layer was dried (Anhyd. Na₂SO₄) and concentrated toobtain the crude product which was purified by Combiflash Rfpurification system.

Compound 254b: By using the above procedure the lipid 254b was isolatedin 72% yield (554 mg). 1H NMR (400 MHz, CDCl3) δ 4.91-4.78 (m, 1H), 4.05(t, J=6.7 Hz, 4H), 3.81 (s, 6H), 3.63 (t, J=6.4 Hz, 1H), 2.29 (dt,J=15.2, 7.5 Hz, 8H), 2.21 (s, 6H), 1.84-1.69 (m, 2H), 1.57 (dt, J=13.4,5.2 Hz, 9H), 1.53-1.40 (m, 4H), 1.27 (s, 43H). 13C NMR (101 MHz, cdcl3)δ 174.45, 174.13, 173.59, 77.54, 77.22, 76.91, 74.34, 64.54, 59.17,51.65, 45.67, 34.56, 34.35, 34.27, 32.67, 29.59, 29.40, 29.33, 29.31,29.25, 28.83, 26.06, 25.51, 25.18, 25.11, 23.38. MW calcd for C₄₃H₇₉NO₁₀770.09; Found 770.68.

Compound 254c: By using the above procedure the lipid 254c was isolatedin 69% (490 mg). 1H NMR (400 MHz, CDCl3) δ 5.71-5.36 (m, 4H), 4.89-4.72(m, 1H), 4.59 (d, J=6.8 Hz, 4H), 2.26 (ddd, J=22.3, 13.0, 8.6 Hz, 9H),2.19 (s, 6H), 2.12-1.95 (m, 4H), 1.82-1.68 (m, 2H), 1.63-1.37 (m, 8H),1.37-1.00 (m, 32H), 0.85 (t, J=6.8 Hz, 6H). 13C NMR (101 MHz, cdcl3) δ173.94, 173.57, 135.61, 123.57, 77.54, 77.22, 76.91, 74.34, 60.40,59.16, 45.65, 34.52, 34.33, 32.66, 31.88, 29.59, 29.57, 29.38, 29.28,29.06, 27.75, 25.49, 25.14, 23.35, 22.81, 14.28. MW calcd for C₄₃H₈₃NO₆:710.12; Found 710.81.

Compound 254d: By using the above procedure the lipid 254d was isolatedin 67% yield (456 mg). 1H NMR (400 MHz, CDCl3) δ 4.92-4.78 (m, 1H), 4.05(t, J=6.7 Hz, 4H), 3.63 (t, J=6.4 Hz, 1H), 2.39-2.24 (m, 8H), 2.21 (s,6H), 1.89-1.70 (m, 2H), 1.69-1.54 (m, 8H), 1.51 (dd, J=17.2, 6.3 Hz,4H), 1.27 (s, 42H), 0.88 (t, J=6.8 Hz, 6H). MW calcd for: C₄₁H₇₉NO₆:682.07; Found 682.96.

Compound 254e: By using the above procedure the lipid 254e was isolatedin 70% (474 mg). 1H NMR (400 MHz, CDCl3) δ 5.49 (ddd, J=12.9, 9.8, 7.3Hz, 2H), 5.40-5.23 (m, 2H), 4.92-4.77 (m, 1H), 4.05 (t, J=6.9 Hz, 4H),2.32 (ddd, J=23.4, 14.5, 7.1 Hz, 12H), 2.21 (s, 6H), 2.07-1.91 (m, 4H),1.84-1.70 (m, 2H), 1.66-1.39 (m, 8H), 1.40-1.15 (m, 26H), 0.88 (t, J=6.8Hz, 5H). MW calc. for C₄₁H₇₅NO₆ (MH⁺): 678.04, found: 678.5.

Compound 254f: By using the above procedure the lipid 254f was isolatedin 73% (559 mg). 1H NMR (400 MHz, CDCl3) δ 5.87-5.62 (m, 2H), 5.55 (dtt,J=9.1, 6.4, 1.3 Hz, 2H), 4.93-4.75 (m, 1H), 4.50 (dd, J=6.5, 0.6 Hz,4H), 2.40-2.17 (m, 13H), 2.12-1.95 (m, 4H), 1.89-1.67 (m, 2H), 1.69-1.44(m, 7H), 1.41-1.12 (m, 25H), 0.88 (t, J=6.9 Hz, 5H). MW calc. forC₄₁H₇₅NO₆ (MH⁺): 678.04, found: 678.5.

Compound 254 g: By using the above procedure the lipid 254g was isolatedin 63% (432 mg). 1H NMR (400 MHz, CDCl3) δ 4.93-4.77 (m, 1H), 4.20-3.95(m, 4H), 2.44-2.23 (m, 8H), 2.21 (s, 6H), 1.84-1.66 (m, 3H), 1.68-1.34(m, 15H), 1.35-1.17 (m, 20H), 1.17-1.04 (m, 5H), 0.88 (dd, J=12.4, 6.6Hz, 16H). MW calcd for C₄₃H₈₃NO₆: 710.12; Found 710.81.

Compound 254h: By using the above procedure the lipid 254h was isolatedin 66% (466 mg). 1H NMR (400 MHz, CDCl3) δ 5.08 (ddd, J=7.1, 5.9, 1.3Hz, 2H), 4.91-4.75 (m, 1H), 4.22-3.97 (m, 4H), 2.39-2.22 (m, 8H), 2.23(d, J=16.7 Hz, 7H), 2.09-1.84 (m, 4H), 1.86-1.71 (m, 3H), 1.71-1.02 (m,44H), 0.91 (t, J=4.9 Hz, 6H). MW calcd for C₄₃H₇₉NO₆: 706.12; Found706.81.

Example 19

In another approach the following synthetic approach is used for thesynthesis of Compound 1 of the present invention.

Example 20

8-benzyloxy-octan-1-ol (2)

To a stirred suspension of NaH (60% in oil, 82 g, 1.7096 mol) in 500 mLanhydrous DMF, a solution of compound 1 (250 g, 1.7096 mol) in 1.5 L DMFwas added slowly with dropping funnel at 0° C. Reaction mixture wasstirred for 30 min and to it Benzyl bromide (208.86 mL, 1.7096 mol) wasadded slowly under nitrogen atmosphere. Reaction was then warmed toambient temperature and stirred for 10 h. After completion of reaction,mixture was quenched with crushed ice (˜2 kg) and extracted with Ethylacetate (2×1 L). Organic layer washed with water (1 L) to removeunwanted DMF, dried over Na₂SO₄ and evaporated to dryness under vacuum.The crude compound was purified on 60-120 silica gel, eluted with 0-5%MeOH in DCM to afford compound 2 (220 g, 54%) as pale yellow liquid. H¹NMR (400 MHz, CDCl₃): δ=7.33-7.24 (m, 5H), 4.49 (s, 2H), 3.63-3.60 (m,2H), 3.47-3.43 (m, 2H), 1.63-1.51 (m, 4H), 1.39-1.23 (m, 8H).

(8-bromo-octyloxymethyl)-benzene (3)

Compound 2 (133 g, 0.5635 mol) was dissolved in 1.5 L of DCM, CBr₄(280.35 g, 0.8456 mol) was added into this stirring solution andreaction mixture was cooled to 0° C. under inert atmosphere. PPh₃(251.03 g, 0.9571 mol) was then added in portions keeping thetemperature below 20° C. and after complete addition reaction wasstirred for 3 h at room temperature and monitored by TLC. Aftercompletion of reaction, solid (PPh₃O) precipitated out from the reactionmixture was filtered off and filtrate was diluted with crushed ice (˜1.5kg) and extracted with DCM (3×750 mL). Organic layer was separated,dried over an. Na₂SO₄ and distilled under vacuum. Resulting crudecompound was chromatographed on 60-120 mesh silica gel column using 0-5%ethyl acetate in hexanes as eluting system to give compound (150 g, 89%)as pale yellow liquid. H¹ NMR (400 MHz, CDCl₃): δ=7.33-7.25 (m, 5H),4.49 (s, 2H), 3.47-3.41 (m, 2H), 3.41-3.37 (m, 2H), 1.86-1.80 (m, 4H),1.62-1.56 (m, 2H), 1.42-1.29 (m, 8H).

1,17-bis-benzyloxy-heptadecan-9-ol (4)

To freshly activated Mg turnings (24.08 g, 1.003 mol) was added 200 mLanhydrous THF was added followed by the addition of pinch of iodine intothe mixture under inert atmosphere. After initiation of the Grignardformation a solution of Compound 3 (150 g, 0.5016 mol) in 1 L of dry THFwas added slowly controlling the exothermic reaction. After completeaddition reaction was refluxed for 1 h and then cooled to roomtemperature. (60.24 g, 1.0033 mol) methyl formate was then added slowlyand reaction was continued for 2 h. After completion, the reaction wasquenched by slow addition of 10% HCl followed by water (1 L) andextracted with Ethyl Acetate (3×1 L). Organic layer was taken in 5 litbeaker, diluted with 500 mL of methanol and cooled to 0° C. To thissolution excess of NaBH₄ (˜5 eq) was added in portions to ensure thehydrolysis of formate ester which was not cleaved by addition of HCl.Resulting solution was stirred for an hour and then volatilites werestripped off under vacuum. Residue was taken in water (1 L) andacidified by 10% HCl solution (P^(H) 4). Product was then extracted outwith ethyl acetate (3×1 L). Organic phase was then dried andconcentrated on rotary evaporator to get the desired compound 4 (57 g,24%) as solid. H¹ NMR (400 MHz, CDCl₃): δ=7.35-7.32 (m, 8H), 7.29-7.24(m, 2H), 4.49 (s, 4H), 3.56 (m, 1H), 3.46-3.43 (m, 4H), 1.63-1.56 (m,4H), 1.44-1.34 (m, 28H). C¹³ NMR (100 MHz, CDCl₃): δ=138.56, 128.21,127.49, 127.34, 72.72, 71.76, 70.37, 37.37, 29.64, 29.56, 29.47, 29.33,26.07, 25.54.

[9-benzyloxy-1-(8-benzylozy-octyl)-nonyloxy]-cert-butyl-dimethyl-silane(5)

Compound 4 (56 g, 0.1196 mol) was dissolved in 700 mL of anhydrous THFand cooled to 0° C. TBMS-Cl (36.06 g, 0.2396 mol) was added slowlyfollowed by addition of Imidazole (32.55 g, 0.4786 mol) under inertatmosphere. Reaction was then stirred at room temperature for 18 h.Reaction was judged complete by TLC and then quenched with ice (˜1 kg)and extracted with Ethyl acetate (3×500 mL). Organic layer wasseparated, washed with Sat NaHCO₃ solution to remove the acidicimpurity, dried over Na₂SO₄ and evaporated under reduce pressure toobtain crude compound which was purified by silica gel (60-120 mesh) andeluted with 0-10% ethyl acetate hexane to yield (60 g, 82%) of compound5 as yellowish oil. H¹ NMR (400 MHz, CDCl₃): δ=7.33-7.24 (m, 10H), 4.49(s, 4H), 3.60-3.57 (m, 1H), 3.46-3.43 (m, 4H), 1.61-1.54 (m, 4H),1.41-1.26 (m, 28H), 0.87 (s, 9H), 0.02 (s, 6H)9-(tert-butyl-dimethyl-silanyloxy)-heptadecane-1,17-diol (6): Compound 5(60 g, 0.1030 mol) was dissolved in 500 mL ethyl acetate and degassedwith N₂ for 20 min. (10 wt %) Pd on carbon (12 g) was added and reactionwas stirred under H₂ atmosphere for 18 h. After completion of reaction(by TLC) mixture was filtered through celite bed and washed with ethylacetate. Filtrate was evaporated under vacuum. The compound 6 (19 g,46%) thus obtained was pure enough to carry out the next reaction. H¹NMR (400 MHz, CDCl₃): δ=3.64-3.58 (m, 5H), 1.59 (br, 2H), 1.57-1.51 (m,4H), 1.38-1.22 (m, 28H), 0.87 (s, 9H), 0.02 (s, 6H).

9-(tert-butyl-dimethyl-silanyloxy)-heptadecanedioic acid (7)

To a stirred solution of 6 (2 g, 0.0049 mol) in anhydrous DMF (40 mL)was added pyridinium dichromate (2.7 g, 0.0074 mol) at 0° C. under inertatmosphere. Reaction mixture was then allowed to warm to roomtemperature over a period of 10-15 minutes and continued for 24 h.Progress of the reaction was monitored by TLC. After complete oxidationreaction was diluted with water (100 mL). Aqueous phase was extractedwith DCM (3×40 mL). Organic phase was washed with brine (1×25 mL) andconcentrated under vacuum to afford crude acid which was then purifiedby (100-200 mesh) silica gel column using 0-30% ethyl acetate in hexanessystem. Pure product 26-003 was obtained (0.7 g, 33%) as pale yellowoil. H¹ NMR (400 MHz, CDCl₃): δ=3.61-3.56 (m, 1H), 2.35-2.32 (m, 4H),1.64-1.59 (m, 4H), 1.40-1.19 (m, 24H), 0.86 (s, 9H), 0.017 (s, 6H);LC-MS [M+H] −431.00; HPLC (ELSD) purity—96.94%

Di((Z)-non-2-en-1-yl) 9-((tert-butyldimethylsilyl)oxy)heptadecanedioate(8)

The diacid 7 (0.42 g, 0.97 mmol) was dissolved in 20 mL ofdichloromethane and to it cis-2-nonen-1-ol (0.35 g, 2.44 mmol) was addedfollowed by Hunig's base (0.68 g, 4.9 mmol) and DMAP (12 mg). To thismixture EDCI (0.47 g, 2.44 mmol) was added and the reaction mixture wasstirred at room temperature overnight and the TLC (silica gel, 5% MeOHin CH₂Cl₂) showed complete disappearance of the starting acid. Thereaction mixture was diluted with CH₂Cl₂ (40 mL) and washed withsaturated NaHCO₃ (50 mL), water (60 mL) and brine (60 mL). The combinedorganic layers were dried over anhyd. Na₂SO₄ and solvents were removedin vacuo. The crude product thus obtained was purified by Combiflash Rfpurification system (40 g silicagel, 0-10% MeOH in CH₂Cl₂) to isolatethe pure product 8 (0.35 g, 53%) as a colorless oil. ¹H NMR (400 MHz,CDCl₃): δ ¹H NMR (400 MHz, CDCl₃) δ 5.64 (dt, J=10.9, 7.4 Hz, 2H),5.58-5.43 (m, 2H), 4.61 (d, J=6.8 Hz, 4H), 3.71-3.48 (m, 1H), 2.30 (t,J=7.6 Hz, 4H), 2.20-1.98 (m, 4H), 1.71-1.53 (m, 4H), 1.31 (ddd, J=8.3,7.0, 3.7 Hz, 34H), 1.07-0.68 (m, 14H), 0.02 (s, 5H). ¹³C NMR (101 MHz,CDCl₃) δ 178.18, 139.81, 127.78, 81.73, 81.42, 81.10, 76.72, 64.59,41.52, 41.32, 38.76, 36.09, 34.10, 33.93, 33.80, 33.70, 33.59, 33.55,33.26, 31.95, 30.34, 29.69, 29.58, 29.39, 27.01, 22.56, 18.48, 0.01.

Di((Z)-non-2-en-1-yl) 9-hydroxyheptadecanedioate (9)

The silyl protected diester 8 (0.3 g, 0.44 mmol) was dissolved in 1 Msolution of TBAF in THF (6 mL) and the solution was kept at 40° C. fortwo days after which the TLC showed the completion of the reaction. Thereaction mixture was diluted with water (60 mL) and extracted with ether(2×50 mL). The combined organic layers were concentrated and the thusobtained crude product was purified by column to isolate the pureproduct (0.097 g, 39%). ¹H NMR (400 MHz, CDCl₃) δ 5.64 (dt, J=10.9, 7.4Hz, 2H), 5.52 (dt, J=11.0, 6.8 Hz, 2H), 4.61 (d, J=6.8 Hz, 4H), 3.57 (s,1H), 2.30 (t, J=7.5 Hz, 4H), 2.09 (q, J=7.1 Hz, 4H), 1.75-1.53 (m, 4H),1.53-1.06 (m, 36H), 0.88 (t, J=6.8 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ173.98, 135.64, 123.57, 77.54, 77.22, 76.91, 72.14, 60.41, 37.69, 34.54,31.89, 29.70, 29.60, 29.44, 29.29, 29.07, 27.76, 25.80, 25.15, 22.82,14.29.

Di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate

The alcohol 9 (0.083 g, 0.147 mmol) was dissolved in 20 mL ofdichloromethane and to it dimethylaminobutyric acid hydrochloride (0.030g, 0.176 mmol) was added followed by Hunig's base (0.045 g, 0.44 mmol)and DMAP (2 mg). To this mixture EDCI (0.034 g, 0.176 mmol) was addedand the reaction mixture was stirred at room temperature overnight andthe TLC (silica gel, 10% MeOH in CH₂Cl₂) showed complete disappearanceof the starting alcohol. The reaction mixture was diluted with CH₂Cl₂(40 mL) and washed with saturated NaHCO₃ (50 mL), water (60 mL) andbrine (60 mL). The combined organic layers were dried over anhyd. Na₂SO₄and solvents were removed in vacuo. The crude product thus obtained waspurified by Combiflash Rf purification system (40 g silicagel, 0-10%MeOH in CH₂Cl₂) to isolate the pure product (0.062 g, 62%) as acolorless oil. ¹H NMR (400 MHz, CDCl₃) δ 5.74-5.58 (m, 2H), 5.51 (dtt,J=9.7, 6.8, 1.3 Hz, 2H), 4.95-4.75 (m, 1H), 4.61 (d, J=6.8 Hz, 4H),2.35-2.24 (m, 8H), 2.22 (d, J=7.9 Hz, 6H), 2.09 (q, J=6.9 Hz, 4H),1.83-1.72 (m, 2H), 1.60 (dd, J=14.4, 7.2 Hz, 4H), 1.49 (d, J=5.7 Hz,4H), 1.41-1.13 (m, 30H), 0.88 (t, J=6.9 Hz, 6H). ¹³C NMR (101 MHz,CDCl₃) δ 173.72, 173.36, 135.40, 123.35, 74.12, 60.18, 58.95, 45.46,34.30, 34.11, 32.45, 31.67, 29.38, 29.35, 29.17, 29.07, 28.84, 27.53,25.28, 24.93, 23.16, 22.59, 14.06. MW calc. for C₄₁H₇₅NO₆ (MH⁺): 678.04,found: 678.5.

In another embodiment the following shorter route was used for thesynthesis of the di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate. The commercial9-bromonon-1-ene 10 was treated with magnesium to form the correspondingGrignard reagent which was reacted with ethylformate to give thecorresponding adduct 11 which on treatment with bromobutyryl chloride toprovide the bromoester 12. The bromoester 12 on treatment with RuO₄provided the diacid 13. The bromodiacid 13 on treatment withdimethylamine provided the amino diacid 14. The aminodiacid 14 oncoupling with the alcohol 15 provided the product in good yields.

Example 21

Example 22

Example 23

Example 24

Compound 501: To a stirred solution of 2-hydroxy 1-octanol 5 g (31.25mmol), DMAP 0.38 g (3.1 mmol) in dry pyridine (100 mL) was added DMTr-Cland stirred at room temperature for 14 h. 10 mL of water was added andextracted with ethyl acetate, washed with saturated NaHCO₃ and brine.The organic layer was dried over Na₂SO₄ and concentration of the solventgave 20 g of crude product 500 which was co-evaporated with toluenetwice and used for the next step without further purification. To theabove crude DMTr ether in dry THF (250 mL) were added NaH and iodomethane at 0° C. and then brought to room temperature over 30 min. andthen stirred for two days. 5 mL of water was added and concentratedfollowed by column chromatography (0-30% ethyl acetate in hexane) gavethe corresponding product 501 (10.25 g, R_(f): 0.45, 20% ethyl acetatein hexane) and 8.4 g of recovered starting material 500. ¹H NMR (400MHz, CDCl₃) δ 7.47-6.8 (m, 13H), 3.79 (s, 6H), 3.42 (s, 3H), 3.29-3.26(m, 1H), 3.13-3.04 (m, 2H), 1.55-1.47 (m, 2H), 1.3-1.2 (m, 10H), 0.89(t, J=6.4 Hz, 3H).

Alcohol 502: The compound 501 (10.25 g, 21.5 mmol) was dissolved in 75mL of 80% acetic acid and stirred at room temperature for 14 h. 10 mL ofmethanol was added and concentrated, followed by column chromatography(0-50% ethyl acetate in hexane) yielded the expected product 502 ascolorless oil (1.8 g, 82%, R_(f): 0.3, 30% ethyl acetate in hexane). ¹HNMR (400 MHz, CDCl₃) δ 3.71-3.65 (m, 1H), 3.5-3.45 (m, 1H), 3.41 (s,3H), 3.28-3.25 (m, 1H), 1.93-1.9 (m, 1H), 1.45-1.41 (m, 2H), 1.39-1.27(m, 10H), 0.88 (s, J=6.8 Hz, 3H).

Compound 503: Compound 503 was synthesized following generalexperimental procedure for compound 213. 0.3 g as pale yellow oil (52%,R_(f)=0.2, 5% methanol in dichloromethane). ¹H NMR (400 MHz, CDCl₃) δ4.87-4.84 (m, 1H), 4.18-4.00 (m, 4H), 3.4 (s, 6H), 3.37-3.19 (m, 2H),2.34-2.26 (m, 6H), 2.2 (s, 6H), 1.8-1.6 (m, 2H), 1.63-1.2 (m, 50H), 0.88(s, J=6.8 Hz, 6H).

Example 25

Compound 504: To a stirred solution of alcohol 11 (4.01 g, 22.25 mmol),TBDPS-Cl (12.24 g, 44.5 mmol) and DMAP (0.54 g, 4.42 mmol) was addedtriethyl amine (8.99 g, 90 mmol) and stirred at room temper for 14 h. Tothe above solution was added imidazole (1.51 g, 22.25 mmol) andcontinued to stir for 14 h at room temperature. 20 mL of water was addedand extracted with DCM followed by washing with 2N HCl, brine and driedover anhydrous Na₂SO₄. Concentration of the solvent gave the crudeproduct which was purified by column chromatography (0-10% ethyl acetatein hexane) to yield compound 504 (7.38 g, 79%, R_(f): 0.8, 5% ethylacetate in hexane). ¹H NMR (400 MHz, CDCl₃) δ 7.68-7.66 (m, 4H),7.43-7.33 (m, 6H), 5.86-5.76 (m, 2H), 5.02-4.91 (m, 4H), 3.73-3.67 (m,1H), 2.04-1.99 (m, 4H), 1.42-1.08 (m, 24H), 1.05 (s, 9H).

Compound 505: To a stirred solution of diene 504 (7.38 g, 17.6 mmol) andRuCl₃ (0.18 g, 0.88 mmol) in 400 mL of DCM/CH₃CN (1:1) was added NaIO₄(37.6 g, 176 mmol) dissolved in 400 mL of water drop wise around 5° C.over 30 min. and stirred at room temperature for 3 h. The organic layerwas separated followed by washing with 3% Na₂S solution (100 mL), water(250 mL) brine and dried over anhydrous Na₂SO₄. Concentration of thesolvent gave the crude product 505 (4 g, 42%, R_(f): 0.3, 40% ethylacetate in hexane), which was used for the next step without furtherpurification.

Compound 506: To a stirred solution of the acid 505 (4 g, 7.22 mmol),HBTU (6.02 g, 15.88 mmol), HOBt (2.14 g, 15.88 mmol) and DMAP (88 mg,0.72 mmol) in 75 mL of dry DCM was added 5 mL of methanol and stirred atroom temperature for 14 h. 10 mL of water was added followed byextraction with DCM (3×50 mL), washing with saturated NaHCO₃, water,brine and dried over anhydrous Na₂SO₄. Concentration of the solvent gavethe crude product which was purified by column chromatography (0-30%ethyl acetate in hexane) to yield compound 506 (2 g, 47.6%, R_(f): 0.3,10% ethyl acetate in hexane). ¹H NMR (400 MHz, CDCl₃) δ 7.67-7.65 (m,4H), 7.41-7.33 (m, 6H), 3.70-3.64 (m, 1H), 3.66 (s, 6H), 2.28 (t, J=7.2Hz, 4H), 1.63-1.07 (m, 24H), 1.04 (s, 9H).

Compound 507: To a stirred solution of dimethyl ester 506 (1.0 g, 1.79mmol) in dry THF (20 mL) were added KHMDS (0.752 g, 3.76 mmol) andmethyl iodide (0.762 g, 5.37 mmol) at 0° C. and then brought to roomtemperature over 30 min. and stirred for 24 h. 10 mL of sat. NH₄Clsolution was added followed by extraction with ethyl acetate (3×50 mL),washing with water, brine and dried over anhydrous Na₂SO₄. Concentrationof the solvent gave the crude product, which was purified by columnchromatography (0-5% ethyl acetate in hexane) to obtain the product 507(0.218 g, 20%, R_(f): 0.8, 5% ethyl acetate in hexane). ¹H NMR (400 MHz,CDCl₃) δ 7.68-7.65 (m, 4H), 7.41-7.33 (m, 6H), 3.70-3.67 (m, 1H), 3.67(s, 6H), 2.43-2.38 (m, 2H), 1.59-1.07 (m, 24H), 1.13 (d, J=7.2 Hz, 6H),1.04 (s, 9H).

Compound 509: To a stirred solution of methyl ester 507 (0.4 g, 0.66mmol) in 10 mL of MeOH/THF (1:1) was added LiOH (0.079 g, 3.27 mmol) in1 mL of water and stirred at room temperature for 24 h. To the abovesolution was added KOH (0.183 g, 3.27 mmol) in 1 mL of water and stirredfor another 2 days. 2 mL of sat. NH₄Cl solution was added followed byextraction with ethyl acetate (3×25 mL), washing with water, brine anddried over anhydrous Na₂SO₄. Concentration of the solvent gave the crudeproduct 508 (0.45 g, R_(f): 0.2, 10% ethyl acetate in hexane), which wasused for the next step without further purification. To a stirredsolution of the above di-acid 508 (0.45 g), cis-2-Nonen-1-ol (0.66 g,4.6 mmol) and EDC.HCl (0.82 g, 4.6 mmol) in dry DCM (15 mL) was addedDIEA (1.2 g, 9.24 mmol) and stirred at room temperature for 3 days. 10mL of water was added followed by extraction with DCM followed bywashing with 2N HCl, brine and dried over anhydrous Na₂SO₄.Concentration of the solvent gave the crude product which was purifiedby column chromatography (0-10% ethyl acetate in hexane) to yieldcompound 509 (0.3 g, 55%, R_(f): 0.5, 3% ethyl acetate in hexane). ¹HNMR (400 MHz, CDCl₃) δ 7.67-7.65 (m, 4H), 7.42-7.33 (m, 6H), 5.67-5.6(m, 2H), 5.55-5.49 (m, 2H), 4.615 (d, J=4 Hz, 4H), 3.71-3.65 (m, 1H),2.44-2.35 (m, 2H), 2.10 (q, J=8.0 Hz, 4H), 1.64-1.07 (m, 40H), 1.13 (d,J=8.0 Hz, 6H), 1.04 (s, 9H), 0.86 (t, J=10 Hz, 6H).

Compound 511: To a stirred solution of silyl ether 509 (0.3 g, 0.36mmol) in dry THF were added pyridine (1 mL) and HF.Pyr., (1 mL) dropwise and stirred at 45° C. for 48 h. The solvent was evaporated and usedfor the next step without purification.

To a stirred solution of the above crude alcohol 510, N,N-Dimethyl aminobutyric acid (0.34 g, 2.04 mmol), EDC.HCl (0.39 g, 2.04 mmol) and DMAP(0.06 g, 0.51 mmol) in dry DCM (10 mL) was added DIEA (0.5 g, 3.88 mmol)and stirred at room temperature for 2 days. 10 mL of water was addedfollowed by extraction with DCM (3×25 mL), washing with saturatedNaHCO₃, water, brine and dried over anhydrous Na₂SO₄. Concentration ofthe solvent gave the crude product which was purified by columnchromatography (0-30% ethyl acetate in 1% TEA containing hexane) toyield compound 511 (0.167 g, 66%, R_(f): 0.4, 10% MeOH in DCM).Molecular weight for C₄₃H₇₉NO₆ (M+H)⁺ Calc. 706.59, Found 706.5.

Example 26

Compound 512: To a stirred solution of 4-Pentynoic acid in 100 mL ofTHF/HMPA (4:1) at −78° C. was added nBuLi (3.1 g, 49 mmol) drop wise andstirred for 30 min. Then the reaction mixture was brought to 0° C. andstirred for 2 h. Again, the reaction mixture was cooled to −78° C. andn-butyl bromide (3.07 g, 22.44 mmol) was added drop wise and stirred atroom temperature for 14 h. 10 mL of sat. NH₄Cl solution was addedfollowed by extraction with ethyl acetate (3×25 mL), washing with water,brine and dried over anhydrous Na₂SO₄. Concentration of the solvent gavethe crude product, which was purified by column chromatography (0-30%ethyl acetate in hexane) to yield compound 512 (0.4 g, R_(f): 0.8, 30%ethyl acetate in hexane). ¹H NMR (400 MHz, CDCl₃) δ 2.59-2.55 (m, 2H),2.49-2.44 (m, 2H), 2.16-2.11 (m, 2H), 1.49-1.34 (m, 4H), 0.9 (t, J=6.0Hz, 3H).

Compound 513: To a suspension of Ni(OAc)₂ (0.45 g, 2.53 mmol) in EtOH(20 mL) was added NaBH₄ (0.096 g, 12.65 mmol) portion wise at roomtemperature and stirred for 15 min. under H₂ atm. Filtered off the solidfollowed by concentration of the solvent gave compound 513 (0.35 g,88.6%, R_(f): 0.6, 20% ethyl acetate in hexane). ¹H NMR (400 MHz, CDCl₃)δ 10.88 (br s, 1H), 5.47-5.41 (m, 1H), 5.35-5.31 (m, 1H), 2.43-2.33 (m,4H), 2.07-2.03 (m, 2H), 1.36-2.27 (m, 4H), 0.9 (t, J=8.0 Hz, 3H).

Compound 515: To a stirred solution of di-acetate 514 (1.5 g, 3.09 mmol)in MeOH (100 mL) was added a piece of sodium metal (0.05 g, 2.17 mmol)and stirred at room temperature for 14 h. Neutralized with dry ice andconcentrated followed by extraction with ethyl acetate (3×50 mL),washing with water, dried over anhydrous Na₂SO₄. Concentration of thesolvent gave the crude product 515 (1.1 g, 88.7%), which was used forthe next step without purification.

Compound 516: To a stirred solution of the above diol 515 (0.4 g, 1mmol), 513 (0.341 g, 2.19 mmol), DMAP (0.1 g, 0.82 mmol) and EDC.HCl(0.57 g, 2.98 mmol) in dry DCM (15 mL) was added DIEA (5.97 g, 6 mmol)and stirred at room temperature for 2 days. 10 mL of water was addedfollowed by extraction with ethyl acetate followed by washing with 1NHCl, brine and dried over anhydrous Na₂SO₄. Concentration of the solventgave the crude product which was purified by column chromatography(0-10% ethyl acetate in hexane) to yield compound 516 (0.335 g, 50%,R_(f): 0.6, 5% ethyl acetate in hexane). ¹H NMR (400 MHz, CDCl₃) δ5.45-5.38 (m, 2H), 5.36-5.29 (m, 2H), 4.06 (t, J=8 Hz, 4H), 3.63-3.58 9m, 1H), 2.39-2.31 (m, 8H), 2.07-2.02 (m, 4H), 1.65-1.57 (m, 4H),1.4-1.28 (m, 32H), 0.9 (t, J=6.0 Hz, 6H), 0.88 (s, 9H), 0.03 (s, 6H).

Compound 517: To a stirred solution of silyl ether 516 (0.3 g, 0.36mmol) in dry THF (5 mL) were added pyridine (1 mL) and HF.Pyr. (1 mL)drop wise and stirred at 45° C. for 24 h. The solvent was evaporatedfollowed by purification by column chromatography gave product 517 (0.2g, 72%, R_(f): 0.4, 10% ethyl acetate in hexane). ¹H NMR (400 MHz,CDCl₃) δ 5.43-5.36 (m, 2H), 5.34-5.27 (m, 2H), 4.04 (t, J=8 Hz, 4H),3.59-3.53 (m, 1H), 2.37-2.3 (m, 8H), 2.05-2.0 (m, 4H), 1.61-1.29 (m,37H), 0.88 (t, J=8.0 Hz, 6H).

Compound 518: To a stirred solution of the alcohol 517 (0.2 g, 0.355mmol), N,N-Dimethyl amino butyric acid (0.36 g, 2.14 mmol), EDC.HCl(0.406 g, 2.14 mmol) and DMAP (0.043 g, 0.36 mmol) in dry DCM (10 mL)was added DIEA (0.55 g, 4.26 mmol) and stirred at room temperature for 2days. 10 mL of water was added followed by extraction with DCM (3×25mL), washing with saturated NaHCO₃, water, brine and dried overanhydrous Na₂SO₄. Concentration of the solvent gave the crude productwhich was purified by column chromatography (0-30% ethyl acetate in 1%TEA containing hexane) to yield compound 518 (0.172 g, 72%, R_(f): 0.2,5% MeOH in DCM). ¹H NMR (400 MHz, CDCl₃) δ 5.43-5.36 (m, 2H), 5.32-5.27(m, 2H), 4.87-4.83 (m, 1H), 4.03 (t, J=6 Hz, 4H), 2.36-2.2 (m, 6H), 2.32(s, 6H), 2.03-1.25 (m, 40H), 0.88 (t, J=6.0 Hz, 6H).

Example 27

Compound 521: To a suspension of Mg in Et₂O was added alkyl bromide (25g, 107.7 mmol) drop wise at 40° C. over one hour. Ethyl formate wasadded to the above reaction mixture at 0-5° C. and then the reactionmixture was stirred at room temperature for 14 h. The reaction mixturewas poured onto the ice cold sat. NH₄Cl solution followed by extractionwith Et₂O (3×250 mL), washing with water, brine and dried over anhydrousNa₂SO₄. Concentration of the solvent gave the crude product, which wasre-dissolved in MeOH (250 mL) and a small piece of sodium (0.1 g) wasadded and stirred at room temperature for 14 h. The solvent wasevaporated and 100 mL of water was added followed by filtration of thesolid, washing with water (2×100 mL) gave pale yellow powder 521 (17 g,94%, %, R_(f): 0.8, 10% ethyl acetate in hexane). ¹H NMR (400 MHz,CDCl₃) δ 5.84-5.74 (m, 2H), 5.0-4.89 (m, 4H), 3.64-3.49 (m, 1H),2.04-1.99 (m, 4H), 1.79 (br s, 1H), 1.44-1.23 (m, 32H).

Compound 522: To a stirred solution of 521 (10 g, 29.73 mmol) and DMAP(0.1 g, 0.82 mmol) in dry DCM (50 mL) was added 4-bromo butyryl chloride(6.56 g, 35.68 mmol) and stirred at room temperature for 14 h. 5 mL ofsaturated NaHCO₃ was added and the organic layer was separated and driedover anhydrous Na₂SO₄. Concentration of the solvent gave the crudeproduct which was purified by column chromatography (0-10% ethyl acetatein hexane) to yield compound 522 (9.6 g, 66.7%, R_(f): 0.9, 5% ethylacetate in hexane).

Compound 524: Oxidation was carried out to get compound 523 (8.6 g,83.5%, R_(f): 0.1, 5% MeOH in DCM) following same experimental procedureas for compound 505. This crude material was dissolved in 2NN,N-dimethyl amine in THF (20 mL) and heated to 60° C. in a sealed tubefor 14 h. Concentrated the reaction mixture and then pH of the reactionmixture was brought to 3. This mixture was freeze-dried to obtaincompound 524 as HCl salt (4 g, 82%). Molecular weight for C27H51NO6(M+H)⁺ Calc. 486.37, Found 486.2. ¹H NMR (400 MHz, CDCl₃) δ 4.94-4.89(m, 1H), 3.32-3.3 (m, 2H), 3.2-3.16 (m, 2H), 2.91 (s, 6H), 2.47 (t, J=8Hz, 2H), 2.28 (t, J=8 Hz, 4H), 2.05-1.97 (m, 2H), 1.61-1.56 (8H),1.4-1.25 (m, 22H).

Synthesis of ester 525, 526, 527 and 528:

The title compounds were synthesized following the experimentalprocedure as for compound 516.

Compound 525: (0.75 g, 60%, R_(f): 0.3, 5% MeOH in DCM). ¹H NMR (400MHz, CDCl₃) δ 5.65-5.59 (m, 2H), 5.53-5.47 (m, 2H), 4.87-4.81 (m, 1H),4.595 (d, J=4.0 Hz, 4H), 2.43-2.25 (m, 8H), 2.2 (s, 6H), 2.1-2.03 (m,4H), 1.81-1.73 (m, 2H), 1.61-1.56 (m, 4H), 1.48-1.47 (m, 4H), 1.36-1.23(m, 32H), 0.86 (t, J=8.0 Hz, 6H).

Compound 526: (0.358 g, 60.9%, R_(f): 0.5, 5% MeOH in DCM). ¹H NMR (400MHz, CDCl₃) δ 4.87-4.81 (m, 1H), 4.07-3.95 (m, 4H), 2.32-2.24 (m, 6H),2.2 (s, 6H), 1.80-1.69 (m, 4H), 1.6-1.14 (m, 46H), 0.88-0.84 (m, 24H).

Compound 527: (0.258 g, 56.8%, R_(f): 0.5, 5% MeOH in DCM). Molecularweight for C47H91NO6 (M+H)⁺ Calc. 766.23; Found: 766.7. ¹H NMR (400 MHz,CDCl₃) δ 4.86-4.80 (m, 1H), 4.12-4.02 (m, 4H), 2.31-2.23 (m, 8H), 2.19(s, 6H), 1.80-1.72 (m, 2H), 1.66-1.06 (m, 52H), 0.87 (d, J=8.0 Hz, 6H),0.84 (d, J=8.0 Hz, 12H).

Compound 528: (0.3 g, 68.1%, R_(f): 0.5, 5% MeOH in DCM). Molecularweight for C47H91NO6 (M+H)⁺ Calc. 766.23; Found: 766.7. ¹H NMR (400 MHz,CDCl₃) δ 4.86-4.80 (m, 1H), 4.12-4.02 (m, 4H), 2.31-2.21 (m, 8H), 2.19(s, 6H), 1.79-1.72 (m, 2H), 1.66-0.98 (m, 52H), 0.87 (d, J=8.0 Hz, 6H),0.835 (d, J=4.0 Hz, 12H).

Example 28

Synthesis of compounds 533, 534, 535 and 536: The title compounds (1mmol) are synthesized following the experimental procedure of compound513 except de-silylation step and it is done using TBAF in THF at roomtemperature.

Synthesis of compounds 537, 538, 539 and 540: The title compounds (1mmol) are synthesized following the experimental procedure of compound525.

Example 29

Compound 243 (X=O/NH, R=alkyl/aryl) can be synthesized as shown inScheme 16-2. Tosyl group of 239 can be replaced with phthalimide groupby nucleophilic substitution. After deprotection followed by couplingwith 111 under acidic conditions, 242 can be synthesized. Standardesterification gives cationic lipid 243 and its analogs.

Example 30 Synthesis of Ester-Containing Lipids

Compound 15: Compound 13 (503 mg, 1.0 mmol) was treated with 14 (469 mg,3.0 mmol) in the presence of EDCI (2.30 g, 12.0 mmol), DMAP (235 mg,1.92 mmol) and DIEA (8.34 mL, 47.9 mmol) in CH₂Cl₂ (50 mL) for 14 h.Aqueous work-up then column chromatography gave compound 15 (1.22 g,1.54 mmol, 40%).

Molecular weight for C₄₉H₉₂NO₆ (M+H)⁺ Calc. 790.6925, Found 790.7.

Compound 16: This compound was synthesized from 13 andtetrahydrolavandulol using a procedure analogous to that described forcompound 15. Yield: 0.358 g, 61%. ¹H NMR (400 MHz, CDCl₃) δ 4.87-4.81(m, 1H), 4.07-3.95 (m, 4H), 2.32-2.24 (m, 6H), 2.2 (s, 6H), 1.80-1.69(m, 4H), 1.6-1.14 (m, 46H), 0.88-0.84 (m, 24H).

Compound 17: This compound was synthesized from 12 (1.0 g, 2.15 mmol)and 3 (1.03 g, 5.16 mmol) using a procedure analogous to that describedfor compound 15.

Yield: 856 mg (50%). ¹H NMR (400 MHz, CDCl₃) δ 4.91-4.79 (m, 1H), 4.08(t, J=7.1 Hz, 4H), 2.35-2.25 (m, 14H), 1.89-1.76 (m, 2H), 1.67-1.13 (m,62H), 0.88 (t, J=7.0 Hz, 12H). ¹³C NMR (100 MHz, CDCl₃) δ 174.08, 74.45,63.08, 45.27, 34.76, 34.56, 34.28, 33.70, 32.61, 32.39, 29.54, 29.36,29.28, 26.36, 25.47, 25.13, 22.83, 14.26. Molecular weight for C₄₉H₉₆NO₆(M+H)⁺ Calc. 794.7238, Found 794.6.

Compound 18: This compound was synthesized from 13 (1.0 g, 2.15 mmol)and 3 (1 g) using a procedure analogous to that described for compound15.

Yield: 1 g (59%). ¹H NMR (400 MHz, CDCl₃) δ 4.94-4.74 (m, 1H), 4.17-3.85(m, 4H), 2.46-2.19 (m, 12H), 1.93-1.79 (m, 2H), 1.74-1.45 (m, 10H), 1.37(d, J=20.2 Hz, 2H), 1.35-1.13 (m, 44H), 0.88 (t, J=6.9 Hz, 12H). ¹³C NMR(101 MHz, CDCl₃) δ 174.19, 77.53, 77.21, 76.90, 63.12, 34.81, 34.66,34.35, 33.76, 32.66, 32.45, 29.76, 29.73, 29.63, 29.48, 29.39, 26.42,25.57, 25.23, 22.89, 14.32. Molecular weight for C₅₃H₁₀₃NO₆ (M+H)⁺ Calc.850.38, Found 850.7.

Compound 19: This compound was synthesized from 12 and 11 using aprocedure analogous to that described for compound 15.

Yield: 860 mg (51%). ¹H NMR (400 MHz, CDCl₃) δ 4.90-4.81 (m, 1H), 4.04(t, J=6.8 Hz, 4H), 2.37-2.17 (m, 14H), 1.84-1.06 (m, 48H), 0.93-0.78 (m,24H). ¹³C NMR (100 MHz, CDCl₃) δ 174.06, 74.35, 65.51, 64.91, 59.05,45.51, 43.77, 37.10, 34.55, 34.29, 32.55, 29.54, 29.37, 29.34, 29.28,28.58, 28.19, 26.99, 26.74, 25.47, 25.15, 22.90, 22.82, 19.60, 19.41,19.28. Molecular weight for C₄₇H₉₂NO₆ (M+H)⁺ Calc. 766.6925, Found766.5.

Compound 20: This compound was synthesized from 13 and 11 using aprocedure analogous to that described for compound 15.

¹H NMR (400 MHz, CDCl₃) δ 4.86 (p, J=6.2 Hz, 1H), 4.04 (t, J=6.7 Hz,4H), 2.38-2.17 (m, 14H), 1.84-1.07 (m, 56H), 0.93-0.76 (m, 24H). ¹³C NMR(100 MHz, CDCl₃) δ 174.11, 173.46, 74.44, 64.90, 59.06, 45.51, 43.77,37.11, 34.59, 34.32, 32.57, 29.71, 29.67, 29.57, 29.43, 29.34, 28.58,28.20, 27.00, 26.75, 25.51, 25.20, 22.90, 22.82, 19.41, 19.28. Molecularweight for C₅₁H₁₀₀NO₆ (M+H)⁺ Calc. 822.7551, Found 822.6.

Compound 21: This compound was synthesized from 12 and 6 using aprocedure analogous to that described for compound 15.

¹H NMR (400 MHz, CDCl₃) δ 4.91-4.78 (m, 1H), 4.15-3.98 (m, 4H),2.39-2.18 (m, 14H), 1.84-1.11 (m, 44H), 0.92-0.77 (m, 24H). ¹³C NMR (100MHz, CDCl₃) δ 174.06, 173.44, 74.36, 63.73, 59.03, 45.48, 41.00, 36.98,34.56, 34.29, 32.54, 29.60, 29.54, 29.49, 29.36, 29.28, 28.52, 25.47,25.13, 23.15, 22.85, 22.81, 19.49, 18.89. Molecular weight for C₄₅H₈₈NO₆(M+H)⁺ Calc. 738.6612, Found 738.6.

Compound 22: This compound was synthesized from 13 and 6 using aprocedure analogous to that described for compound 15.

Yield: 900 mg (57%). ¹H NMR (400 MHz, CDCl₃) δ 4.92-4.78 (m, 1H),4.15-3.91 (m, 4H), 3.33-3.08 (m, 1H), 2.36-2.15 (m, 14H), 1.79 (dq,J=14.3, 7.2 Hz, 2H), 1.74-1.55 (m, 8H), 1.55-1.37 (m, 9H), 1.35-0.95 (m,36H), 0.96-0.61 (m, 27H). ¹³C NMR (101 MHz CDCl₃) δ 174.16, 173.52,77.54, 77.22, 76.91, 74.48, 63.76, 59.10, 45.55, 42.02, 41.04, 38.75,37.09, 37.02, 34.65, 34.36, 32.62, 30.71, 29.75, 29.72, 29.64, 29.62,29.53, 29.48, 29.44, 29.38, 28.56, 28.45, 25.56, 25.23, 23.59, 23.23,22.90, 22.86, 19.54, 19.03, 18.94. Molecular weight for C₄₉H₉₅NO₆ (M+H)⁺Calc. 794.2817, Found 794.7.

Compound 24: This compound was synthesized from 13 and 23 using aprocedure analogous to that described for compound 15.

Yield: 0.567 g (30%). ¹H NMR (400 MHz, CDCl₃) δ 4.85 (p, J=6.1 Hz, 1H),4.20-3.93 (m, 4H), 2.41-2.18 (m, 13H), 1.92-1.72 (m, 2H), 1.56 (ddd,J=27.4, 16.4, 5.8 Hz, 12H), 1.39 (s, 2H), 1.25 (s, 54H), 0.91 (dt,J=13.7, 6.4 Hz, 11H). ¹³C NMR (101 MHz, CDCl₃) δ 174.18, 173.51, 77.54,77.23, 76.91, 74.50, 63.12, 59.10, 45.55, 34.81, 34.66, 34.38, 33.76,32.67, 32.62, 32.45, 29.77, 29.73, 29.64, 29.49, 29.39, 26.42, 25.57,25.24, 23.23, 22.89, 14.32. Molecular weight for C₄₇H₈₈NO₆ (M+H)⁺ Calc.762.6612, Found 762.5.

Example 31 Synthesis of Alcohol Components

Compound 2: Compound 2 was synthesized from 1 using a procedureanalogous to that described in Journal of the Organic Chemistry, 2009,1473.

¹H NMR (400 MHz, CDCl₃) δ 3.66 (s, 3H), 2.23 (d, J=6.9 Hz, 2H), 1.84(brs, 1H), 1.27 (d, J=11.5 Hz, 16H), 0.88 (t, J=6.8 Hz, 6H). ¹³C NMR(100 MHz, CDCl₃) δ 174.29, 51.49, 39.25, 35.22, 34.00, 32.24, 26.34,22.77, 14.22.

Compound 3: To a suspension of LiAlH₄ (2.84 g, 74.9 mmol) in THF (85 mL)was added a solution of compound 2 (8.55 g, 37.4 mmol) in THF (25 mL).The reaction mixture was refluxed overnight. Aqueous workup then columnchromatography gave pure compound 3 (7.35 g, 36.7 mmol, 98%) as acolorless oil.

¹H NMR (400 MHz, CDCl₃) δ 3.66 (t, J=7.0 Hz, 2H), 1.59-1.12 (m, 19H),0.88 (t, J=6.9 Hz, 6H).

Compound 4: Tetrahydrolavandulol (10.1 g, 63.8 mmol) was treated withmethansulfonyl chloride (6.38 mL) in CH₂Cl₂ (200 mL) and Et₃N (17.6 mL).Aqueous workup gave the crude mesylate, which was treated with KCN (4.98g, 76.5 mmol) in EtOH (90 mL) and H₂O (10 mL). Aqueous workup thencolumn chromatography gave pure compound 4 (8.36 g, 50.0 mmol, 72%) as acolorless oil.

¹H NMR (400 MHz, CDCl₃) δ 2.38-2.23 (m, 2H), 1.86-1.78 (m, 1H),1.59-1.42 (m, 3H), 1.40-1.07 (m, 3H), 0.93-0.89 (m, 12H). ¹³C NMR (100MHz, CDCl₃) δ 119.73, 41.69, 36.46, 30.10, 28.44, 28.33, 22.82, 22.59,19.62, 19.11, 19.05.

Compound 6: The cyano derivative 4 was converted to the ethyl esterunder acidic conditions to give compound 5 and the ester was reduced byLiAlH₄ in THF to give compound 6.

Compound 7: Tetrahydrolavandulol (98.1 g, 51.2 mmol) was oxidized withPCC (16.6 g, 76.8 mmol) in CH₂Cl₂ (200 mL). Aqueous workup then columnchromatography gave pure compound 7 (6.19 g, 39.6 mmol, 77%) as acolorless oil.

¹H NMR (400 MHz, CDCl₃) δ 9.60 (d, J=3.1 Hz, 1H), 2.05-1.79 (m, 1H),1.71-1.36 (m, 4H), 1.23-1.04 (m, 2H), 1.02-0.82 (m, 12H).

Compound 9: To a solution of compound 7 (2.0 g, 12.8 mmol) in toluene(40 mL) and CH₂Cl₂ (18 mL) and was added 8 (3.96 g, 11.8 mmol). Themixture was heated at 70° C. overnight. Column chromatography gave purecompound 9 (1.40 g, 6.59 mmol, 51%) as a colorless oil.

¹H NMR (400 MHz, CDCl₃) δ 6.77 (dd, J=15.6, 9.9 Hz, 1H), 5.76 (d, J=15.6Hz, 1H), 3.73 (s, 3H), 1.97-1.83 (m, 1H), 1.72-1.64 (m, 1H), 1.54-1.40(m, 2H), 1.37-1.22 (m, 1H), 1.18-0.97 (m, 2H), 0.94-0.78 (m, 12H). ¹³CNMR (100 MHz, CDCl₃) δ 167.19, 152.54, 121.70, 51.53, 49.66, 36.95,31.76, 29.49, 28.29, 22.92, 22.54, 20.84, 19.24.

Compound 10: To a solution of compound 9 (1.0 g, 4.71 mmol) in MeOH (15mL) was added Pd—C (125 mg). The mixture was stirred under H₂ atmosphereovernight. The mixture was filtered over Celite then evaporated to givepure compound 10 (924 mg, 4.31 mmol, 92%) as a colorless oil.

¹H NMR (400 MHz, CDCl₃) δ 3.67 (s, 3H), 2.41-2.16 (m, 2H), 1.74-1.57 (m,2H), 1.57-1.42 (m, 2H), 1.33-1.02 (m, 5H), 0.88-0.83 (m, 12H). ¹³C NMR(100 MHz, CDCl₃) δ 174.78, 51.62, 43.71, 36.97, 32.69, 29.23, 28.56,27.94, 25.92, 22.85, 22.79, 19.32, 19.19.

Compound 11: To a suspension of LiAlH₄ (444 mg, 11.7 mmol) in THF (12mL) was added a solution of compound 10 (1.25 g, 5.83 mmol) in THF (8mL). The reaction mixture was refluxed overnight. Aqueous workup gavethe crude compound 11 (1.1 g) as a colorless oil.

¹H NMR (400 MHz, CDCl₃) δ 3.63 (t, J=6.7 Hz, 2H), 1.74-1.66 (m, 1H),1.60-1.45 (m, 3H), 1.37-1.05 (m, 7H), 0.88-0.82 (m, 12H). ¹³C NMR (100MHz, CDCl₃) δ 63.75, 44.00, 37.16, 31.22, 29.40, 28.61, 28.28, 26.62,22.90, 22.82, 19.43, 19.28.

Example 32 Synthesis of Ester-Containing Lipids

Compound 26: Compound 25 (840 mg, 1.03 mmol) was stirred in TFA (9 mL)and CH₂Cl₂ (36 mL) for 3 h at room temperature. Evaporation of thesolvents and co-evaporation with toluene 3 times gave compound 26.

Molecular weight for C₄₃H₈₀NO₆ (M+H)⁺ Calc. 706.5986, Found 706.4.

Compound 27: Compound 26 from the previous step was treated with2,2-dimethylpropanol (363 mg, 4.12 mmol) in the presence of EDCI (592mg, 3.09 mmol), DMAP (50 mg, 0.412 mmol) and DIEA (1.44 mL, 8.24 mmol)in CH₂Cl₂ (10 mL) for 14 h. Aqueous work-up then column chromatographygave compound 27 (575 mg, 0.679 mmol, 66%).

¹H NMR (400 MHz, CDCl₃) δ 5.40-5.28 (m, 4H), 4.91-4.81 (m, 1H), 3.76 (s,4H), 2.34-2.27 (m, 8H), 2.22 (s, 6H), 2.03-1.97 (m, 8H), 1.83-1.26 (m,50H), 0.94 (s, 18H). ¹³C NMR (100 MHz, CDCl₃) δ 174.14, 173.53, 130.09,129.92, 74.41, 73.72, 59.12, 45.61, 34.60, 34.32, 32.64, 31.45, 29.93,29.85, 29.71, 29.68, 29.48, 29.32, 29.28, 27.39, 27.33, 26.62, 25.52,25.22, 23.32.

Molecular weight for C₅₃H₁₀₀NO₆ (M+H)⁺ Calc. 846.7551, Found 846.5.

Example 33 Synthesis of Quaternary Lipids

A. The amino lipids synthesized in Examples 31 and 32 can be convertedto the corresponding quaternary lipids as shown below by treatment withCH₃Cl in CH₃CN and CHCl₃.

B. Synthesis of BODIPY-lipid conjugates

Compound 102: To a solution of compound 101 (2.00 g, 4.30 mmol) andcis-2-nonen-1-ol (1.81 mL, 10.7 mmol) in CH₂Cl₂ (20 mL) were addeddiisopropylethylamine (3.00 mL, 17.2 mmol),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (2.06 g,10.7 mmol) and DMAP (106 mg, 0.868 mmol). The reaction mixture wasstirred at room temperature for 18 hours. The reaction mixture wasdiluted with CH₂Cl₂ (200 mL) and washed with saturated NaHCO₃ aq. (100mL). The organic layer was dried over MgSO₄, filtered and concentrated.The crude was purified by silica gel column chromatography (0-5% EtOAcin Hexane) to give compound 102 (2.11 g, 2.96 mmol, 69%, R_(f)=0.45developed with 10% EtOAc in Hexane).

¹H NMR (500 MHz, CDCl₃) δ 5.67-5.61 (m, 2H), 5.54-5.49 (m, 2H),4.89-4.84 (m, 1H), 4.62 (d, J=6.5 Hz, 4H), 3.46 (t, J=6.5 Hz, 2H), 2.48(t, J=7.3 Hz, 2H), 2.30 (t, J=7.5 Hz, 4H), 2.20-2.14 (m, 2H), 2.12-2.04(m, 4H), 1.63-1.60 (m, 4H), 1.51-1.50 (m, 4H), 1.37-1.27 (m, 32H), 0.88(t, J=6.8 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 173.90, 172.45, 135.58,123.51, 74.74, 60.36, 34.47, 34.24, 32.93, 32.91, 31.83, 29.54, 29.48,29.31, 29.21, 29.01, 28.03, 27.70, 25.43, 25.08, 22.76, 14.23.

Molecular weight for C₃₉H₆₉BrNaO₆ (M+Na)⁺ Calc. 735.42, Found 735.2.

Compound 103: To a solution of 102 (2.11 g, 2.96 mmol) in DMF (20 mL)was added a solution of N-Boc-1,6-diaminohexane (670 mg, 3.10 mmol) inDMF (20 mL) at 0° C. The mixture was stirred for 18 hours at roomtemperature. Then additional N-Boc-1,6-diaminohexane (160 mg, 0.740mmol) in DMF (1 mL) was added and the mixture was stirred for 12 hour.The reaction was quenched by adding saturated NaHCO₃ aq. (100 mL) thenextracted with Et₂O (150 mL×3). The organic layer was separated anddried over anhydrous MgSO₄. After filtration and concentration, thecrude was purified by silica gel column chromatography (5% MeOH inCH₂Cl₂, R_(f)=0.24) to give 103 (1.28 g, 1.51 mmol, 51%).

¹H NMR (400 MHz, CDCl₃) δ 5.67-5.61 (m, 2H), 5.55-5.50 (m, 2H),4.88-4.81 (m, 1H), 4.61 (d, J=6.8 Hz, 4H), 4.54 (brs, 1H), 3.11-3.08 (m,2H), 2.67-2.59 (m, 4H), 2.35 (t, J=7.4 Hz, 2H), 2.29 (t, J=7.6 Hz, 4H),2.10-2.07 (m, 4H), 1.84-1.81 (m, 4H), 1.63-1.57 (m, 4H) 1.50-1.47 (m,8H), 1.44 (s, 9H), 1.38-1.27 (m, 34H), 0.88 (t, J=6.8 Hz, 6H). ¹³C NMR(100 MHz, CDCl₃) δ 173.90, 173.53, 135.57, 123.50, 74.49, 60.36, 49.82,49.29, 40.64, 34.47, 34.24, 32.68, 31.83, 30.16, 29.89, 29.54, 29.50,29.33, 29.23, 29.01, 28.58, 27.69, 27.11, 26.80, 25.44, 25.37, 25.09,22.76, 14.23.

Molecular weight for C₅₀H₉₃N₂O₈ (M+H)⁺ Calc. 849.69, Found 849.5.

Compound 104: To a solution of 103 (1.16 g, 1.37 mmol) in THF (20 mL)were added formaldehye (37 wt. % in H₂O, 0.306 mL, 4.11 mmol), sodiumcyanoborohydride (1 M solution in THF, 2.06 mL, 2.06 mmol) and aceticacid (0.008 mL, 0.137 mmol) at 0° C. The mixture was stirred at roomtemperature for 17 hours. The reaction was quenched by adding saturatedNaHCO₃ aq. (50 mL) then extracted with Et₂O (100 mL×3). The organiclayer was separated and dried over anhydrous MgSO₄. After filtration andconcentration, the crude was purified by silica gel columnchromatography (8% MeOH in CH₂Cl₂, R_(f)=0.46) to give 104 (531 mg,0.615 mmol, 45%).

¹H NMR (400 MHz, CDCl₃) δ 5.66-5.60 (m, 2H), 5.53-5.47 (m, 2H),4.86-4.80 (m, 1H), 4.61-4.59 (m, 5H), 3.12-3.07 (m, 2H), 2.89-2.78 (m,4H), 2.62 (s, 3H), 2.40 (t, J=6.8 Hz, 2H), 2.28 (t, J=7.4 Hz, 4H),2.11-2.06 (m, 4H), 1.99-1.92 (m, 2H), 1.69-1.27 (m, 57H), 0.87 (t, J=6.8Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 173.86, 172.45, 156.18, 135.55,123.45, 75.24, 60.32, 56.68, 55.83, 40.72, 40.36, 34.40, 34.09, 31.79,31.29, 29.92, 29.49, 29.41, 29.26, 29.17, 28.96, 28.55, 27.65, 26.49,26.30, 25.41, 25.02, 24.79, 22.71, 20.12, 14.19.

Molecular weight for C₅₁H₉₅N₂O₈ (M+H)⁺ Calc. 863.71, Found 863.6.

Compound 105: To a solution of compound 104 (525 mg, 0.608 mmol) inCH₂Cl₂ (8 mL) was added trifluoroacetic acid (2 mL) at 0° C. Thereaction mixture was stirred at 0° C. for 1 hour and at room temperaturefor 3 hours. The reaction mixture was evaporated and co-evaporated withtoluene 3 times then dried in vacuo overnight to give compound 105 (603mg, 0.603 mmol calculated as 2 TFA salt, quantitatively, R_(f)=0.24developed with 8% MeOH in CH₂Cl₂).

¹H NMR (400 MHz, CDCl₃) δ 8.06 (brs, 1H), 5.68-5.61 (m, 2H), 5.55-5.49(m, 2H), 4.87-4.81 (m, 1H), 4.62 (d, J=6.8 Hz, 4H), 4.28 (brs, 3H),3.20-3.02 (m, 6H), 2.82 (d, J=4.0 Hz, 3H), 2.45-2.40 (m, 2H), 2.30 (t,J=7.4 Hz, 4H), 2.12-2.00 (m, 6H), 1.78-1.22 (m, 52H), 0.88 (t, J=6.8 Hz,6H). ¹³C NMR (100 MHz, CDCl₃) δ 174.04, 172.08, 161.84, 161.47, 135.63,123.44, 117.60, 114.71, 75.56, 60.41, 55.69, 55.27, 39.94, 39.64, 34.44,34.06, 31.82, 30.72, 29.53, 29.43, 29.28, 29.19, 29.00, 27.69, 26.58,25.42, 25.27, 25.05, 24.60, 23.06, 22.75, 19.00, 14.22.

Molecular weight for C₄₆H₈₇N₂O₆ (M+H)⁺ Calc. 763.66, Found 763.4.

Compound 106: To a solution of 105 (23.8 mg, 0.0240 mmol, calculated as2TFA salt) in CH₂Cl₂ (1 mL) and Et₃N (0.050 mL, 0.360 mmol) was added asolution of BODIPY® 493/503 (10 mg, 0.0240 mmol, Life Technology #D2191)in CH₂Cl₂ (2 mL). The reaction mixture was stirred for 1 h. The reactionmixture was loaded onto silica gel column chromatography and eluted with0-5% MeOH in CH₂Cl₂. The product color fractions were collected (5% MeOHin CH₂Cl₂, R_(f)=0.36) to give 106 (26 mg, 0.024 mmol, quantitatively).

¹H NMR (400 MHz, CDCl₃) δ 6.05 (s, 2H), 5.67-5.61 (m, 2H), 5.54-5.48 (m,2H), 4.85-4.82 (m, 1H), 4.61 (d, J=6.8 Hz, 4H), 3.37-3.32 (m, 2H),3.27-3.22 (m, 2H), 2.51-2.44 (m, 17H), 2.34-2.27 (m, 8H), 2.12-2.06 (m,4H), 1.60-1.21 (m, 52H), 0.88 (t, J=6.8 Hz, 6H).

Molecular weight for C₆₂H₁₀₄BF₂N₄O₇ (M+H)⁺ Calc. 1065.80, Found 1065.5.

Example 34 Multi-Ester Containing Lipids and Acetal Linked Lipids

Synthesis of compound 5002: To a stirred solution of alcohol 5001 (1.0g, 5.15 mmol), Glycolic anhydride 5000 (5.66 mmol) in DCM (20 mL) wasadded DMAP (1.26 g, 10.41 mmol) and stirred at room temperature for 48h. The reaction mixture was concentrated followed by column purificationgave the corresponding product 5002 (1.4 g, 86%) as DMAP salt. LCMS:Calculated: 316.22 (M⁺), Found: 315.1 (M⁺−1).

Synthesis of compound 5004: To a stirred solution of alcohol 5003 (5.0g, 44.6 mmol), 4-(Dimethylamino)butyric acid hydrochloride (8.1 g, 48.3mmol) and EDC (10.3 g, 53.6 mmol) in DCM (100 mL) was added DIEPA (23 g,178.3 mmol) and stirred at room temperature overnight. After usual workup, the crude product was purified by column chromatography (9.0 g,90%).

Synthesis of compound 5005: To a stirred solution of diene 5004 (4.0 g,18 mmol) in 10 mL of THF was added 9-BBN and stirred overnight. To theabove solution was added 6.6 mL of 3M NaOAc and 7.4 mL of 30% H₂O₂ at0-5° C. The reaction mixture was stirred at room temperature overnight.After usual work up, the crude material was purified by columnchromatography to get 5005 (2.6 g, 55%) as viscous oil. LCMS:Calculated: 261.19 (M⁺), Found: 262.1 (M⁺+1).

Synthesis of compound 5006 and 5007: To a stirred solution of diene 5005(260 mg, 1 mmol), acid 5002 (1.0 g, 2.28 mmol), EDC (387 mg, 2 mmol) in10 mL of DCM was added DIEA (516 mg, 4 mmol) and stirred overnight.After usual work up, the crude material was purified by columnchromatography to get 5006 (0.1 g, 12%) and 5007 (0.2 g, 36%). LCMS forcompound 5006: Calculated: 857.62 (M⁺), Found: 858.5 (M⁺+1), 880.5(M⁺+Na). LCMS for compound 5007: Calculated: 559.4 (M⁺), Found: 560.4(M⁺+1).

Synthesis of compound 5011: To a stirred solution of alcohol 5008 (2.66g 10 mmol) in 5 mL of Chlorotrimethylsilane was added paraformaldehyde(0.3 g, 10 mmol) and stirred at room temperature overnight. The excessChlorotrimethylsilane was evaporated followed by drying under reducedpressure gave the corresponding product 5009 and used for next stepwithout purification. The compound 5009 was added dropwise to thesolution of diol (261 mg, 1 mmol), DIEA (2.5 g, 19.4 mmol) and DMAP (20mg, 0.16 mmol) in DCM (10 mL) and stirred overnight. Concentration ofthe solvent gave the crude product 5010, which was dissolved in 5 mL ofTHF and 2 mL of 1N NaOH was added and stirred for 2 days at roomtemperature. After usual work up, the crude material was purified bycolumn chromatography to get the corresponding product 5011 (200 mg,28%). LCMS for compound 5010: Calculated: 1131.95 (M⁺), Found: 1096.98(M⁺−Cl)). LCMS for compound 5011: Calculated: 704.63 (M⁺), Found: 727.5(M⁺+Na).

Synthesis of compound 5012: To a stirred solution of alcohol 5011 (200mg, 0.284 mmol), 4-(Dimethylamino)butyric acid hydrochloride (103 mg,0.57 mmol), EDC (109 mg, 0.57 mmol) in 10 mL of DCM was added DIEA (294mg, 4 mmol) and stirred overnight. After usual work up, the crudematerial was purified by column chromatography to get 5012 (190 mg,85%). LCMS for compound 5012: Calculated: 817.72 (M⁺), Found: 818.5(M⁺+Na).

Synthesis of compound 5016: To a stirred solution of alcohol 5013 (1.0 g7.03 mmol) in 5 mL of Chlorotrimethylsilane was added acetaldehyde (0.3g, 7.03 mmol) and stirred at room temperature for 2 h. The excessChlorotrimethylsilane was evaporated followed by drying under reducedpressure gave the corresponding product 5014 and used for next stepwithout purification. The compound 5014 was added dropwise to thesolution of diol 5015 (223 mg, 0.55 mmol), DIEA (2 mL g, 11.5 mmol) andDMAP (20 mg, 0.16 mmol) in DCM (10 mL) and stirred overnight. 10 mL ofwater was added followed by extraction with DCM (3×30 mL), washed withwater, saturated NaHCO₃, brine and dried over anhydrous Na₂SO₄.Concentration of the solvent gave the crude product, which was used forthe next step without purification. LCMS for compound 5016: Calculated:738.66 (M⁺), Found: 761.5 (M⁺+Na).

Synthesis of compound 5017: To a stirred solution of alcohol 5016 in 5mL of THF was added 0.54 mL of 1M TBAF in THF (0.54 mmol) and stirredfor 2 days at room temperature. After usual work up, the crude materialwas purified by column chromatography to get 5017. However, it containssome inseparable impurity and hence used for next step without furtherpurification. LCMS for compound 5017: Calculated: 624.57 (M⁺), Found:647.5 (M⁺+Na).

Synthesis of compound 5018: To a stirred solution of alcohol 5017 (0.55mmol), 4-(Dimethylamino)butyric acid hydrochloride (116 mg, 0.64 mmol),EDC (123 mg, 0.64 mmol) in 10 mL of DCM was added DIEA (165 mg, 1.28mmol) and stirred for 2 days. After usual work up, the crude material ispurified by column chromatography (0-10% MeOH in 1% Et₃N containing DCM)to get 5018 (300 mg, 75% from 5015). LCMS for compound 5018: Calculated:737.65 (M⁺), Found: 738.6 (M⁺+1), 760.5 (M⁺+Na⁺).

Example 35 Preparation of Lipid Nanoparticles

The cationic lipids described herein are used to formulate liposomescontaining the AD-1661 duplex (shown in the table below) using anin-line mixing method as described in International Publication No. WO2010/088537, which is incorporated by reference in its entirety. Thelipid nanoparticles had the formulation shown in the table below.

Mole Percentage (Based on 100% of the lipid Component components in theLNP) Cationic lipid 50% Distearoylphosphatidylcholine (DSPC) 10%Cholesterol 38.5%  1-(monomethoxy-polyethyleneglycol)- 1.5% 2,3-dimyristoylglycerol (PEG-DMG) (with an average PEG molecular weightof 2000) siRNA (AD-1661) —

The siRNA AD-1661 duplex has the sequence shown below.

SEQ ID Duplex Sequence 5′-3′ NO: Target AD-1661 GGAfUfCAfUfCfUfCAAGfUfCf1 FVII UfUAfCdTsdT GfUAAGAfCfUfUGAGAfUGAfUf 2 CfCdTsdT

Lower case is 2′OMe modification and Nf is a 2′F modified nucleobase, dTis deoxythymidine, s is phosphothioate

The lipid nanoparticles was prepared as follows. Cationic lipid, DSPC,cholesterol, and PEG-DMG in the ratio recited in the table above weresolubilized in ethanol at a total lipid concentration of 25 mg/mL.

A siRNA stock solution was prepared by solubilizing the siRNA AD-1661 ina low pH acetate or citrate buffer (pH=4) at 0.8 mg/mL.

The stock solutions should be completely clear and the lipids should becompletely solubilized before combining with the siRNA. Therefore, if itwas determined appropriate, the stock solutions were heated tocompletely solubilize the lipids.

The individual stock solutions were combined by pumping each solution toa T-junction (i.e., by in-line mixing). Specifically, the ethanolsolution (at 5 ml/min, via 0.01 in. PEEK tube) and aqueous buffersolution (at 15 mL/min, via 0.02 in. PEEK tube) were mixed through aT-junction (PEEK Tee body, IDEX).

After the T-junction a single tubing is placed where the combined streamwill emit. Ethanol is removed and exchanged for PBS by dialysis. Thelipid formulations are then concentrated using centrifugation ordiafiltration to an appropriate working concentration.

Lipid nanoparticles containing the cationic lipids listed in the tablein Example 36 were prepared as described above.

Example 36 Efficacy of Lipid Nanoparticles

Factor VII (FVII), a prominent protein in the coagulation cascade, issynthesized in the liver (hepatocytes) and secreted into the plasma.FVII levels in plasma can be determined by a simple, plate-basedcolorimetric assay. As such, FVII represents a convenient model fordetermining siRNA-mediated downregulation of hepatocyte-derivedproteins.

Test formulations of the lipid nanoparticles prepared in Example 35 wereinitially assessed for their FVII knockdown in female 7-9 week old,15-25 g, female C57Bl/6 mice at 0.1, 0.3, 1.0 and 5.0 mg/kg with 3 miceper treatment group. All studies included animals receiving eitherphosphate-buffered saline (PBS, control group) or a benchmarkformulation. Formulations were diluted to the appropriate concentrationin PBS immediately prior to testing. Mice were weighed and theappropriate dosing volumes calculated (10 μl/g body weight). Test andbenchmark formulations as well as PBS (for control animals) wereadministered intravenously via the lateral tail vein. Animals wereanesthetised 24 hours later with an intraperitoneal injection ofketamine/xylazine and 500-700 μl of blood was collected by cardiacpuncture into serum separator tubes (BD Microtainer). Blood wascentrifuged at 2,000×g for 10 minutes at 15° C. and serum was collectedand stored at −70° C. until analysis. Serum samples were thawed at 37°C. for 30 minutes, diluted in PBS and aliquoted into 96-well assayplates. Factor VII levels were assessed using a chromogenic assay(Biophen FVII kit, Hyphen BioMed) according to the manufacturer'sinstructions and absorbance was measured in a microplate reader equippedwith a 405 nm wavelength filter. Plasma FVII levels were quantified andED₅₀ values (dose resulting in a 50% reduction in plasma FVII levelscompared to control animals) were calculated using a standard curvegenerated from a pooled sample of serum from control animals. Thoseformulations of interest showing high levels of FVII knockdown(ED₅₀<<0.1 mg/kg) were re-tested in independent studies at a lower doserange to confirm potency and establish ED₅₀ levels.

The following table shows ED₅₀ values for some of the cationic lipidsdescribed herein. Two asterisks (**) indicates an ED₅₀ value between0.001 and 0.10. One asterisk (*) indicates an ED₅₀ value greater than0.10.

ED₅₀ Cationic Lipid **

**

**

**

**

**

**

**

*

**

*

**

**

**

**

**

**

**

**

**

**

**

**

**

**

**

Example 37 Hydrophobicity and Stability

The log P values for the biodegrabable cationic lipids listed in thetable below were calculated using the software available athttp://www.molinspiration.com/services/logp.html from MolinspirationCheminformatics of Slovensky Grob, Slovak Republic.

Furthermore, the HPLC retention time for each biodegradable cationiclipid was measured in lipid nanoparticles prepared from them. The lipidnanoparticles were prepared as described in Example 35 using AD-1661 asthe payload. The retention times are reported in the table belowrelative to the retention time for cholesterol.

The HPLC buffer used was a mixture of two solutions (Solution #1 andSolution #2).

-   -   Solution #1: 80% methanol/20% 10 mM NH₄HCO₃    -   Solution #2: 80% methanol/20% isopropanol

The ratios of the two solutions in the mixture changed over time asindicated in the table below.

Time Solution #1 Solution #2 (min) (vol %) (vol %) 0 70 30 4 10 90 6 1090 6.1 70 30 8 70 30

The size of the lipid nanoparticles was measured before and afterundergoing dialysis overnight. In general, greater changes in lipidnanoparticle size are indicative of lesser stability.

Dynamic laser light scattering was used to determine the lipidnanoparticle size (expressed as the intensity weighted diameter) with aZetasizer (Malvern Instruments, Inc. of Westborough, Mass.). Allmeasurements were made at 532 nm wavelength at the scattering angle of173° using normal resolution mode as the analysis model.

The results of these experiments are provided in the table below.

t(lipid)- LNPs Size Cationic Lipid logP t(chol) (nm) change

9.647 −1.4 107 −> 260

9.972 0.848 73 −> 77

10.093 1.44 60 −> 67

10.201 1.751 59 −> 60

10.259 2.106

10.313 2.365 56 −> 56

10.315 2.219 68 −> 67

10.416 2.707

10.495 3.178

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1. A compound of the formula:

or a salt thereof, wherein R′ is absent, hydrogen, or C₁-C₄ alkyl; withrespect to R¹ and R², (i) R¹ and R² are each, independently, optionallysubstituted alkyl, alkenyl, alkynyl, cycloalkylalkyl, heterocycle, orR¹⁰; (ii) R¹ and R², together with the nitrogen atom to which they areattached, form an optionally substituted heterocylic ring; or (iii) oneof R¹ and R² is optionally substituted alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkylalkyl, or heterocycle, and the other forms a 4-10member heterocyclic ring or heteroaryl with (a) the adjacent nitrogenatom and (b) the (R)_(a) group adjacent to the nitrogen atom; eachoccurrence of R is, independently, —(CR³R⁴)—; each occurrence of R³ andR⁴ are, independently H, halogen, OH, alkyl, alkoxy, —NH₂, R¹⁰,alkylamino, or dialkylamino; each occurrence of R¹⁰ is independentlyselected from PEG and polymers based on poly(oxazoline), poly(ethyleneoxide), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone),poly[N-(2-hydroxypropyl)methacrylamide] and poly(amino acid)s, wherein(i) the PEG or polymer is linear or branched, (ii) the PEG or polymer ispolymerized by n subunits, (iii) n is a number-averaged degree ofpolymerization between 10 and 200 units, and (iv) the compound offormula (III) has at most two R¹⁰ groups; the dashed line to Q is absentor a bond; when the dashed line to Q is absent then Q is absent or is—O—, —NH—, —S—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)N(R⁴)—, —N(R⁵)C(O)—,—S—S—, —OC(O)O—, —O—N═C(R⁵)—, —C(R⁵)═N—O—, —OC(O)N(R⁵)—,—N(R⁵)C(O)N(R⁵)—, —N(R⁵)C(O)O—, —C(O)S—, —C(S)O— or —C(R⁵)═N—O—C(O)—; orwhen the dashed line to Q is a bond then (i) b is 0 and (ii) Q and thetertiary carbon adjacent to it (C*) form a substituted or unsubstituted,mono- or bi-cyclic heterocyclic group having from 5 to 10 ring atoms;each occurrence of R⁵ is, independently, H or C₁-C₄ alkyl; M¹ and M² areeach, independently, a biodegradable group; each occurrence of R^(z) is,independently, C₁-C₈ alkyl; a is 1, 2, 3, 4, 5 or 6; b is 0, 1, 2, or 3;L¹ and L² are each, independently, C₁-C₅ alkylene or C₂-C₅ alkenylene; Xand Y are each, independently, C₄-C₂₀ alkylene or C₄-C₂₀ alkenylene; andZ¹ and Z² are each, independently, C₈-C₁₄ alkyl or C₈-C₁₄ alkenyl,wherein the alkenyl group may optionally be substituted with one or twofluorine atoms at the alpha position to a double bond which is betweenthe double bond and the terminus of Z¹ or Z², and with the proviso thatthe terminus of at least one of Z¹ and Z² is separated from the group M¹or M² by at least 8 carbon atoms.
 2. The compound of claim 1, wherein L¹and L² are each —CH₂—.
 3. The compound of claim 1, wherein L¹ and L² areeach —(CH₂)₂— or —(CH₂)₃—.
 4. The compound of claim 1, wherein X and Yare each, independently —(CH₂)_(n) wherein n is 4, 5, 6, 7 or
 8. 5. Thecompound of claim 4, wherein X and Y are —(CH₂)₇—, —(CH₂)₈—, or—(CH₂)₉—.
 6. A compound of the formula:

or a salt thereof, wherein R′ is absent, hydrogen, or C₁-C₄ alkyl; withrespect to R¹ and R², (i) R¹ and R² are each, independently, optionallysubstituted alkyl, alkenyl, alkynyl, cycloalkylalkyl, heterocycle, orR¹⁰; (ii) R¹ and R², together with the nitrogen atom to which they areattached, form an optionally substituted heterocylic ring; or (iii) oneof R¹ and R² is optionally substituted alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkylalkyl, or heterocycle, and the other forms a 4-10member heterocyclic ring or heteroaryl with (a) the adjacent nitrogenatom and (b) the (R)_(a) group adjacent to the nitrogen atom; eachoccurrence of R is, independently, —(CR³R⁴)—; each occurrence of R³ andR⁴ are, independently H, halogen, OH, alkyl, alkoxy, —NH₂, R¹⁰,alkylamino, or dialkylamino; each occurrence of R¹⁰ is independentlyselected from PEG and polymers based on poly(oxazoline), poly(ethyleneoxide), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone),poly[N-(2-hydroxypropyl)methacrylamide] and poly(amino acid)s, wherein(i) the PEG or polymer is linear or branched, (ii) the PEG or polymer ispolymerized by n subunits, (iii) n is a number-averaged degree ofpolymerization between 10 and 200 units, and (iv) wherein the compoundof formula (IIIA) has at most two R¹⁰ groups; the dashed line to Q isabsent or a bond; when the dashed line to Q is absent then Q is absentor is —O—, —NH—, —S—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)N(R⁴)—,—N(R⁵)C(O)—, —S—S—, —OC(O)O—, —O—N═C(R⁵)—, —C(R⁵)═N—O—, —OC(O)N(R⁵)—,—N(R⁵)C(O)N(R⁵)—, —N(R⁵)C(O)O—, —C(O)S—, —C(S)O— or —C(R⁵)═N—O—C(O)—; orwhen the dashed line to Q is a bond then (i) b is 0 and (ii) Q and thetertiary carbon adjacent to it (C*) form a substituted or unsubstituted,mono- or bi-cyclic heterocyclic group having from 5 to 10 ring atoms;each occurrence of R⁵ is, independently, H or C₁-C₄ alkyl; M¹ and M² areeach, independently, a biodegradable group; each occurrence of R^(z) is,independently, C₁-C₈ alkyl; a is 1, 2, 3, 4, 5 or 6; b is 0, 1, 2, or 3;each R^(z) is, independently, C₁-C₈ alkyl; L¹ and L² are each,independently, C₁-C₅ alkylene or C₂-C₅ alkenylene; X and Y are each,independently, C₄-C₂₀ alkylene or C₄-C₂₀ alkenylene; and Z¹ and Z² areeach, independently, C₁-C₈ alkyl or C₂-C₈ alkenyl; wherein said compoundis not one selected from:


7. The compound of claim 6, wherein L¹ and L² are each —(CH₂)₂—.
 8. Thecompound of claim 6, wherein L¹ and L² are each —(CH₂)₃—.
 9. Thecompound of claim 6, wherein X and Y are each, independently,—(CH₂)_(n)— wherein n is 7-9.
 10. The compound of claim 6, wherein M¹and M² are —C(O)O— (where the carbonyl group in M¹ and M² is bound tothe variable X, and the oxygen atom in M¹ and M² is bound to thevariable L¹ and L²).
 11. The compound of claim 6, wherein theR′R¹R²N—(R)_(a)-Q-(R)_(b)— group is selected from (CH₃)₂N—(CH₂)₃—C(O)O—,(CH₃)₂N—(CH₂)₂—NH—C(O)O—, (CH₃)₂N—(CH₂)₂—OC(O)—NH—, and(CH₃)₂N—(CH₂)₃—C(CH₃)═N—O—.
 12. The compound of claim 6, wherein Z¹, Z²,and each R^(z) are C₃-C₈ alkyl.
 13. The compound of claim 12, whereinZ¹, Z², and each R^(z) are C₃-C₆ alkyl.
 14. A compound of the formula:

or a salt thereof, wherein R′ is absent, hydrogen, or C₁-C₄ alkyl; withrespect to R¹ and R², (i) R¹ and R² are each, independently, optionallysubstituted alkyl, alkenyl, alkynyl, cycloalkylalkyl, heterocycle, orR¹⁰; (ii) R¹ and R², together with the nitrogen atom to which they areattached, form an optionally substituted heterocylic ring; or (iii) oneof R¹ and R² is optionally substituted alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkylalkyl, or heterocycle, and the other forms a 4-10member heterocyclic ring or heteroaryl with (a) the adjacent nitrogenatom and (b) the (R)_(a) group adjacent to the nitrogen atom; eachoccurrence of R is, independently, —(CR³R⁴)—; each occurrence of R³ andR⁴ are, independently H, halogen, OH, alkyl, alkoxy, —NH₂, R¹⁰,alkylamino, or dialkylamino; each occurrence of R¹⁰ is independentlyselected from PEG and polymers based on poly(oxazoline), poly(ethyleneoxide), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone),poly[N-(2-hydroxypropyl)methacrylamide] and poly(amino acid)s, wherein(i) the PEG or polymer is linear or branched, (ii) the PEG or polymer ispolymerized by n subunits, (iii) n is a number-averaged degree ofpolymerization between 10 and 200 units, and (iv) the compound (IV) offormula has at most two R¹⁰ groups; the dashed line to Q is absent or abond; when the dashed line to Q is absent then Q is absent or is —O—,—NH—, —S—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)N(R⁴)—, —N(R⁵)C(O)—, —S—S—,—OC(O)O—, —O—N═C(R⁵)—, —C(R⁵)═N—O—, —OC(O)N(R⁵)—, —N(R⁵)C(O)N(R⁵)—,—N(R⁵)C(O)O—, —C(O)S—, —C(S)O— or —C(R⁵)═N—O—C(O)—; or when the dashedline to Q is a bond then (i) b is 0 and (ii) Q and the tertiary carbonadjacent to it (C*) form a substituted or unsubstituted, mono- orbi-cyclic heterocyclic group having from 5 to 10 ring atoms; eachoccurrence of R⁵ is, independently, H or C₁-C₄ alkyl; M¹ and M² areeach, independently, a biodegradable group; each occurrence of R^(z) is,independently, C₁-C₈ alkyl; a is 1, 2, 3, 4, 5 or 6; b is 0, 1, 2, or 3;L¹ and L² and are each, independently, C₁-C₅ alkylene or C₂-C₅alkenylene; X and Y are each, independently, alkylene or alkenylene; andeach occurrence of Z is independently C₁-C₄ alkyl.
 15. The compound ofclaim 14, wherein X and Y are C₁₂-C₂₀ alkylene or C₁₂-C₂₀ alkenylene.16. The compound of claim 14, wherein -L¹-C(Z)₃ is —CH₂C(CH₃)₃ or—CH₂CH₂C(CH₃)₃.
 17. A cationic lipid or a salt thereof having: (i) acentral carbon atom, (ii) a nitrogen containing head group directlybound to the central carbon atom, and (iii) two hydrophobic tailsdirectly bound to the central carbon atom, wherein each hydrophobic tailis of the formula —R^(e)-M-R^(f) where R^(e) is a C₄-C₁₄ alkyl oralkenyl, M is a biodegradable group, and R^(f) is a branched alkyl oralkenyl, such that (i) the chain length of —R^(e)-M-R^(f) is at most 20atoms, and (ii) the group —R^(e)-M-R^(f) has at least 20 carbon atoms,wherein the alkyl or alkenyl group in R^(e) is optionally substitutedwith one or two fluorine atoms at the alpha position to the M¹ or M²group; and the alkenyl group in R^(f) is optionally substituted with oneor two fluorine atoms at the alpha position to a double bond which isbetween the double bond and the terminus of R^(f).
 18. (canceled) 19.(canceled)
 20. The compound of claim 1, wherein the compound is in theform of a pharmaceutically acceptable salt.
 21. The compound of claim 1,wherein the compound is in the form of a cationic lipid.
 22. A lipidparticle comprising a neutral lipid, a lipid capable of reducingaggregation, and a cationic lipid of claim
 21. 23. The lipid particle ofclaim 22, wherein the neutral lipid is selected from DSPC, DPPC, POPC,DOPE, or SM; the lipid capable of reducing aggregation is a PEG lipid;and the lipid particle further comprises a sterol.
 24. The lipidparticle of claim 22, wherein the cationic lipid is present in a molepercentage of about 20% and about 60%; the neutral lipid is present in amole percentage of about 5% to about 25%; the sterol is present in amole percentage of about 25% to about 55%; and the PEG lipid is PEG-DMA,PEG-DMG, or a combination thereof, and is present in a mole percentageof about 0.5% to about 15%.
 25. The lipid particle of claim 22, whereinthe lipid capable of reducing aggregation is PEG-DMG.
 26. The lipidparticle of claim 25, wherein the lipid particle comprises about 50 mole% of the cationic lipid, about 10% DSPC, about 38.5% cholesterol, andabout 1.5% PEG-DMG (based on 100% of the lipid components in the lipidparticle).
 27. The lipid particle of claim 22, further comprising anactive agent.
 28. The lipid particle of claim 27, wherein the activeagent is a nucleic acid selected from a plasmid, an immunostimulatoryoligonucleotide, an siRNA, an antisense oligonucleotide, a microRNA, anantagomir, an aptamer, and a ribozyme.
 29. A pharmaceutical compositioncomprising a lipid particle of claim 22 and a pharmaceuticallyacceptable carrier.
 30. A method of modulating the expression of atarget gene in a cell, comprising providing to the cell a lipid particleof claim
 22. 31. The method of claim 30, wherein the active agent is anucleic acid is an siRNA.
 32. A method of treating a disease or disordercharacterized by the overexpression of a polypeptide in a subject,comprising providing to the subject the pharmaceutical composition ofclaim 29, wherein the active agent is a nucleic acid selected from thegroup consisting of an siRNA, a microRNA, and an antisenseoligonucleotide, and wherein the siRNA, microRNA, or antisenseoligonucleotide includes a polynucleotide that specifically binds to apolynucleotide that encodes the polypeptide, or a complement thereof.33. A method of treating a disease or disorder characterized byunderexpression of a polypeptide in a subject, comprising providing tothe subject the pharmaceutical composition of claim 29, wherein theactive agent is a plasmid that encodes the polypeptide or a functionalvariant or fragment thereof.
 34. A method of inducing an immune responsein a subject, comprising providing to the subject the pharmaceuticalcomposition of claim 29, wherein the active agent is animmunostimulatory oligonucleotide.